The Interrelationships of Rotylenchulus Reniformis Linford

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The Interrelationships of Rotylenchulus Reniformis Linford and The Interrelationships of Rotylenchulus Reniformis Linford and

Oliveira With Rhizoctonia Solani Kuhn on Cotton. Oliveira With Rhizoctonia Solani Kuhn on Cotton.

Ambalavanan Sankaralingam Louisiana State University and Agricultural & Mechanical College

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The interrelationships o f Rotylenchulus reniformis Linford and Oliveira w ith Rhizoctonia solani K uhn on cotton

Sankaralingam, Ambalavanan, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1993

U M I300 N. ZeebRd.Ann Arbor, MI 48106

THE INTERRELATIONSHIPS OF ROTYLENCHULUS RENIFORMIS LINFORD AND OLIVEIRA WITH RHIZOCTONIA SOLANI KUHN ON COTTON

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Doctor of Philosophy

in

The Department of Plant Pathology and Crop Physiology

byAmbalavanan Sankaralingam

B. Sc. in Agriculture, Annamalai University, India, 1978 M. Sc. in Agriculture, Tamil Nadu Agricultural University, India, 1980

May, 1993

ACKNOWLEDGEMENTS

I express my sincere thanks to my major professor and advisor Dr. E. C. McGawley for

the guidance and encouragement rendered throughout the period of my doctoral studies. I

would also like to appreciate him for all the personal help he provided to me. I would like to thank

equally Dr. W. H. Patrick, Jr. , Director, Wetland Biogeochemistry Institute, L. S. U. , for

sponsoring my studies with a Louisiana Methodist World Hunger Scholarship.

I am grateful to the members of my committee Dr. J. P. Snow, Dr. L. L. Black, Dr. R. W.

Schneider, Dr. M. C. Rush, Dr. J. S. Russin, and Dr. C. Overstreet for the suggestions and

support given during my period of study and research.

I thank all my friends, especially Kanagasabapathi Sathasivan, Lori Grelen, Elizabeth

Higgins, Richard Miller, Pat Hives, Ida Wenefrida, Mark Jones, Randall Johnson, and Paul

Chambers who have given all the possible help during the course of my Ph. D. program.

I thank my parents Mr. and Mrs. Ambalavanan and in-laws Mr. and Mrs. Balasubramanian

for their moral support. I wish to express my deep love to my wife Muthulakshmi and daughters

Gomathi and Ulaganayaki for their patience, affection and care.

I would like to dedicate this dissertation to my late sister.

TABLE OF CONTENTS

pageACKNOWLEDGEMENTS..................................................................................................... ii

LIST OF TABLES................................................................................................................... iv

LIST OF FIGURES................................................................................................................ vi

ABSTRACT............................................................................................................................... vii

CHAPTER1 REVIEW OF LITERATURE......................................................................... 1

Literature Cited............................................................................................ 5

2 THE INTERRELATIONSHIPS OF ROTYLENCHULUS RENIFORMISWITH RHIZOCTONIA SOLANI................................................................. 9Introduction................................................................................................... 10Materials and Methods............................................................................... 11Results................... 14Discussion..................................................................................................... 29Summary ................................................................................... 35Literature Cited ....................................................................................... 37

3 INFLUENCE OF RHIZOCTONIA SOLANI ON EGG HATCHING ANDINFECTIVITY OF ROTYLENCHULUS RENIFORMIS............................ 40Introduction................................................................................................... 41Materials and Methods.............................................................................. 41Results.......................................................................................................... 45Discussion.................................................................................................... 52Summary...................................................................................................... 56Literature Cited........................................................................................... 57

4 CONCLUSIONS............................................................................................ 59Areas for Future Research.......................................................................... 61

VITA........................................................................................................................................... 62

iii

LIST OF TABLES

Table 2.1. Population density of Rotylenchulus reniformis on three cotton cultivars at 90 days after inoculation............................................................................................................... 15

Table 2.2. Effect of Rotylenchulus reniformis on fresh weights of three cotton cultivars at 90 days after inoculation...................................................................................................... 17

Table 2.3. Effects of Rhizoctonia solani isolates on Rotylenchulus reniformis populations on Deltapine 90 cotton at 40 days after inoculation........................................................18

Table 2.4. Effects of Rotylenchulus reniformis and Rhizoctonia solani isolates on fresh weights and disease indices of Deltapine 90 cotton at 40 days after inoculation...19

Table 2.5. Effects of Rhizoctonia solani isolates and cotton cultivars on density of two populations of Rotylenchulus reniformis, at 40 days after inoculation.................... 20

Table 2.6. Effects of Rotylenchulus reniformis populations, Rhizoctonia solani isolates and cultivars on cotton fresh weights and disease indices at 40 days after inoculation.........................................................................................................................23

Table 2.7. Effects of nematode inoculum levels, Rhizoctonia solani isolates and cotton cultivars on population density of Rotylenchulus reniformis at 60 days after inoculation.......................................................................................................................... 24

Table 2.8. Effects of inoculum levels of Rotylenchulus reniformis and Rhizoctonia solani isolates on fresh weights and disease indices of two cotton cultivars at 60 days after inoculation..........................................................................................................................26

Table 2.9. Effects of nematode inoculum levels and Rhizoctonia solani on population density of Rotylenchulus reniformis on Deltapine 90 cotton at 90 days after inoculation...27

Table 2.10. Effects of inoculum levels of Rotylenchulus reniformis and Rhizoctonia solani on fresh weights and disease indices of Deltapine 90 cotton at 90 days after inoculation..........................................................................................................................28

Table 2.11. Effects of nematode inoculum levels, Rhizoctonia solani isolates and cotton cultivars on population density of Rotylenchulus reniformis at 90 days after inoculation..........................................................................................................................31

Table 2.12. Effects of inoculum levels of Rotylenchulus reniformis, Rhizoctonia solani isolates and cotton cultivars on fresh weights and disease indices at 90 days after inoculation..........................................................................................................................32

Table 3.1. Response of eggs of Rotylenchulus reniformis to culture filtrates of Rhizoctonia solani............................. 46

Table 3.2. Response of eggs of Rotylenchulus reniformis to Rhizoctonia solani and growth medium.......................... 48

Table 3.3. Response of eggs of Rotylenchulus reniformis to filtrates of Rhizoctonia solani produced over water culture in compartmentalized “ I plates ”............................... 50

Table 3.4. Hatching of eggs of Rotylenchulus reniformis as influenced by root exudates ofDeltapine 90 cotton seedlings inoculated and noninoculated with Rhizoctonia solani............................ 51

Table 3.5. Influence of Rhizoctonia solani infection on infectivity of Rotylenchulus reniformis on Deltapine 90 cotton............................................................................... 53

Table 3.6. Influence of Rhizoctonia solani infection and hypocotyl wounding on infectivity of Rotylenchulus reniformis on Deltapine 90 cotton......................................................53

LIST OF FIGURES

Fig. 2.1. Means for the juveniles of Rotylenchulus reniformis recovered from soil in experiment 3, for the interaction between Rhizoctonia solani and cotton cultivar. Vertical lines delimit standard errors of means. * = lsol.1 = Isolate 1, Isol. 2 = Isolate 2....................22

Fig. 2.2. Means for plant fresh weights for the interaction between Rhizoctonia solani and Rotylenchulus reniformis in experiment 5. Vertical lines delimit standard errors of means. *-F = absence of fungus. +F = presence of fungus. **0, 500, 2,000 and 8,000 nematodes/pot, respectively............................................................................................. 30

Fig. 3.1. Means for the cumulative egg hatch of Rotylenchulus reniformis in experiment 2, for the interaction between medium x fungus. Vertical lines delimit standard errors of means. *-F = absence of fungus. +F = presence of fungus. **PDB = potato dextrose broth. SDDW = sterile deionized distilled water.............................................49

vi

ABSTRACT

The interrelationships between the reniform nematode, Rotylenchulus reniformis

Linford and Oliveira, and the fungus Rhizoctonia solani Kuhn were studied on cotton

(Gossypium hirsutum L.). Parasitism of cotton seedlings by R. solani resulted in enhanced

reproduction of R. reniformis. In the presence of the fungus, there was increased egg

production by the nematode which resulted in augmented soil population density. Enhanced

reproduction of the nematode in the presence of the fungus was consistent across isolates of

R. solani, populations and inoculum levels of R. reniformis, and cotton cultivars. The nematode

did not influence the severity of seedling blight disease. At 90 days, when R. solani and R.

reniformis were present together, effects on cotton growth were antagonistic.

Culture filtrates of R. solani obtained from potato dextrose broth were inhibitory to the

hatching of eggs of R. reniformis, and the medium was more inhibitory than the filtrate. Filtrates

of R. solani collected from sterile deionized distilled water did not affect egg hatching. Root

exudates from cotton seedlings increased the hatching of eggs of R. reniformis. Exudates from

the root systems of R. solani infected and noninfected cotton seedlings did not differ in their

effect on egg hatching. However, infectivity of preadult females to cotton seedlings was

significantly augmented in the presence of Rhizoctonia solani.

vii

CHAPTER 1

REVIEW OF LITERATURE

1

Nematodes abound in the humid semitropical environment of Louisiana where they

cause injury to the root systems of most agriculturally important plant species. Moreover, their

role in altering the incidence or severity of diseases caused by soilborne plant pathogens,

especially fungi, is well documented in the phytopathological literature (1.2, 1.10, 1.15, 1.24,

1.32, 1.33, 1.37-1.39, 1.41, 1.44).

Rotylenchulus reniformis Linford and Oliveira, commonly known as the reniform

nematode because of the shape of the adult female body, was first described from cowpea in

1940 (1.25). Reniform nematode is an obligate parasite with only females parasitizing plant

roots. There are four molts in the life cycle of the nematode. The first molt takes place within the

egg and the second stage juvenile hatches and undergoes three successive molts to become a

male or a preadult female. The preadult female is the infective stage. Preadults feed as semi-

endoparasites, attain the reniform shape within 4-5 days of infection, and start depositing eggs

within a matrix 3-4 days later. The entire life cycle is completed within 17-25 days depending on

the host and the nematode can develop from juvenile to male or preadult female without

feeding. R. reniformis is known to parasitize 65 plant species belonging to 30 different plant

families (1.26). It was first identified as a pathogen of cotton in Georgia by Smith and Taylor in

1940 (1.43) and was first associated with cotton failure in Louisiana by Birchfield and Jones in

1961 (1.4).

Among the 16 major cotton producing states of the United States, Louisiana ranked 5th

in 1991 with production of 1.4 million bales (1.1). Cotton occupies a major cultivated area in

Louisiana covering 0.33 million hectares and accounting for 21.9% of the gross farm income

(1.1). Yield loss in cotton due to diseases was estimated at 10% (1.3) in Louisiana. Cotton yield

loss attributable to nematodes was estimated at 4% (1.3) and reniform nematode is probably

responsible for much of the damage in Louisiana. (E. C. McGawley and C. Overstreet, pers.

comm.). To date, this nematode has been reported from 47 of the 64 parishes in Louisiana

parasitizing a wide variety of plants.

Seedling diseases caused by fungi such as Rhizoctonia solani Kuhn, Pythium spp., and

Fusarium spp. accounted for an annual loss in 1991 in cotton, ranging from a low of 0.7% in New

Mexico to a high of 9% in Tennessee with Louisiana averaging 2% (1.3). In 1958, Sinclair

reported R. solani to be the fungus most frequently isolated from diseased cotton seedlings in

Louisiana (1.42) and this statement remains accurate in 1992 (K. L. Whitam, Louisiana

Cooperative Extension Service, pers. comm.). This fungus occurs in all parishes in the state;

and, like R. reniformis, its host range is extensive. Soil samples from cotton fields containing

plants exhibiting seedling blight symptoms due to R. solani commonly contain R. reniformis ( C.

Overstreet, pers. comm.). Moreover, the density of R. reniformis populations in soil is usually

higher in areas where seedling disease occurs compared with areas where disease symptoms

are not apparent.

Many investigators have reported nematode related increases in the incidence of

seedling disease caused by R. solani. Increase in the incidence of seedling blight of cotton in

the presence of the root knot nematode, Meloidogyne incognita (Kofoid & White) Chitwood,

was first reported by Reynolds and Hanson (1.37). Synergistic effects on the severity of

seedling disease were found between R. solani and Meloidogyne spp. in cotton (1.6-1.9,1.45),

tomato (Lycopersicon esculentum Mill.) (1.11,1.28,1.29), french bean (Phaseolus vulgaris L.)

(1.36), gram (Cicer arietinum L.) (1.35), tobacco (Nicotiana tabacum L.) (1.31), and radish

(Raphanus sativus L.) (1.21). In the presence of R. solani, resistance to M. incognita in pepper

(Capsicum annuum L.) and tomato is lost (1.12, 1.13). In sugarbeet (Beta vulgaris L.), the

presence of Heterodera schachtii Schmidt resulted in enhanced penetration of seedlings by R.

solani (1.30). In contrast, when R. solani and M. javanica (Treub) Chitwood were present

together, the numbers of nematode-induced galls were significantly decreased in cowpea

(Vigna sinensis Endl.) (1.16,1.17).

Plant pathogenic fungi can also have significant influences on nematode reproduction.

R. solani was antagonistic to the multiplication of M. incognita and R. reniformis in cowpea (1.19,

1.20). Culture filtrates of this fungus inhibited hatching of eggs of M. javanica (1.27, 1.34).

Addition of culture filtrate of R. solani to soil reduced the populations of R. reniformis, M.

incognita and Hoplolaimus indicus (Sher.) compared to nontreated controls in tomato (1.14).

The ratio of male to female of Heterodera rostochiensis Wollenweber increased in tomato when

R. solani was present (1.18).

Rotylenchulus reniformis and its interrelationships with R. solani have been studied by

only a few investigators. Lambe and Wendell reported the presence of R. reniformis and R.

solani together in cotton in Texas (1.23). Brodie and Cooper (1.5) were the first to investigate

the interrelationships of reniform nematode and R. solani on cotton and demonstrated that at

20,000 nematodes/500 gram of soil, susceptibility to postemergence damping-off was greater

than that which occurred in the absence of the nematode. In India, Kumar and Sivakumar found

that when R. reniformis and R. solani were present, okra (Abelmoschus esculentus L.) plants

succumbed to wilt earlier than that which was observed with R. solani alone (1.22). When okra

plants were inoculated with R. solani and R. reniformis water absorption and plant growth were

less than that which was found with separate inoculations (1.40). Although a few reports (1.5,

1.23) have noted the association of R. reniformis and R. solani on cotton, an in-depth study of

their interrelationship has not been conducted. There are gaps in our knowledge as it relates to

the ecology and root pathology of R. reniformis and R. solani on major crop plants including

cotton. Specific objectives of this study were 1) to evaluate the impact of R. solani on

reproduction of R. reniformis, 2) to monitor the influence of R. reniformis on the severity of

Rhizoctonia seedling blight of cotton, 3) to study the effect of infestation with both pathogens

on cotton growth, 4) to evaluate the influence of culture filtrates of R. solani and exudates from

R. solani infected cotton seedlings on the hatching of eggs of R. reniformis, and infectivity of

preadults.

5

Literature Cited

1.1. Anonymous, 1991. Louisiana Summary, Agriculture and Natural Resources, Louisiana Cooperative Extension Service. Louisiana State University Agricultural Center. 14 pp.

1.2. Atkinson, G. F. 1892. Some diseases of cotton. Bulletin of Alabama Agricultural Experiment Station 46:61-65.

1.3. Blasingame, D. 1992. Cotton Disease Loss Estimate Committee report. Pp.165 in D. J. Herber and D A. Richter, eds. Proceedings of Beltwide Cotton Conferences, vol. 1. Memphis, TN: National Cotton Council.

1.4. Birchfield, W., and J. E. Jones. 1961. Distribution of the reniform nematode in relation to crop failure of cotton in Louisiana. Plant Disease Reporter 45:671-673.

1.5. Brodie, B. B., and W. E. Cooper. 1964. Relation of parasitic nematodes to postemergence damping-off of cotton. Phytopathology 54:1023-1027.

1.6. Carter, W. W. 1975. Effect of soil temperatures and inoculum levels of Meloidogyne incognita and Rhizoctonia solani on seedling disease of cotton. Journal of Nematology 7:229-233.

1.7. Carter, W. W. 1975. Effect of soil texture on the interaction between Rhizoctonia solani and Meloidogyne incognita on cotton seedlings. Journal of Nematology 7:234-236.

1.8. Carter, W. W. 1981. The effect of Meloidogyne incognita and tissue wounding on severity of seedling disease of cotton caused by Rhizoctonia solani. Journal of Nematology 13:374-376.

1.9. Cauquil, J. E., and R. L. Shepherd. 1970. Effect of root knot nematode fungi combinations on cotton seedling disease. Phytopathology 60:448-451.

1.10. Chahal, P. P. K., and H. K. Chhabra. 1984. Interaction of Meloidogyne incognita with Rhizoctonia solani on tomato. Indian Journal of Nematology 16:56-57.

1.11. Chahal, P. P. K., and H. K. Chhabra. 1984. Effect of Meloidogyne incognita and Rhizoctonia solani on the emergence and damping-off of tomato seedlings. Journal of Research of the Punjab Agricultural University 21:642-644.

1.12. Hasan, A. 1985. Breaking resistance in chilli to root knot nematode by fungal pathogens. Nematologica 31:210-217.

1.13. Hasan, A., and M. N. Khan. 1985. The effect of Rhizoctonia solani, Sclerotium rolfsii and Verticillium dahliae on the resistance of tomato to Meloidogyne incognita. Nematologia Mediterranea 13:133-136.

1.14. Haseeb, A., and M. M. Alam. 1984. Soil population of plant parasitic nematodes infecting tomato in relation to metabolites of Rhizoctonia solani. Indian Journal of Plant Pathology 2:189-190.

6

1.15. Inagaki, H., and N. T. Powell. 1969. Influence of the root lesion nematode on black shank symptom development in flue-cured tobacco. Phytopathology 59:1350-1355.

1.16. Kanwar, R. S., D. C., Gupta, and K. K. Walia. 1987. Interaction of Meloidogyne javanica and Rhizoctonia solani on cowpea. Nematologia Mediterranea 15:385-386.

1.17. Kanwar, R. S., D. C., Gupta, and K. K. Walia. 1989. Effect of nematode inoculations at different intervals and soil types on interactions between Meloidogyne javanica and Rhizoctonia solani on cowpea. Indian Journal of Nematology 18:112-113.

1.18. Ketudat, U. 1969. The effect of some soil-borne fungi on the sex ratio of Heterodera rostochiensis on tomato. Nematologica 15:229-233.

1.19. Khan, T. A., and S. I. Husain. 1988. Effect of individual, concomitant and sequential inoculations of Rhizobium, Rotylenchulus reniformis, Meloidogyne incognita and Rhizoctonia solani on cowpea plant growth, disease development and nematode multiplication. Indian Journal of Nematology 18:232-238.

1.20. Khan, T. A., and S. I. Husain. 1990. Studies on the effect of interactions of variable inoculum levels of Rotylenchulus reniformis, Meloidogyne incognita and Rhizoctonia solani on cowpea. Current Nematology 1:49-52.

1.21. Khan, M. V., and J. Muller. 1986. Interaction between Rhizoctonia solani and Meloidogyne hapla on radish in gnotobiotic culture. Libyan Journal of Agriculture 16:137- 140.

1.22. Kumar, S., and C. V. Sivakumar. 1981. Disease complex involving Rotylenchulus reniformis and Meloidogyne incognita on okra. Nematologia Mediterranea 9:145-149.

1.23. Lambe, R. C., and W. Horne. 1963. The reniform nematode in cotton in the lower Rio Grand valley of Texas. Plant Disease Reporter 47:941.

1.24. LaMondia, J. A., and S. B. Martin. 1989. The influence of Pratylenchus penetrans and temperature on black root rot of strawberry by binucleate Rhizoctonia spp. Plant Disease 73:107-110.

1.25. Linford, M. B., and Juliette M. Oliveira. 1940. Rotylenchulus reniformis, Nov. gen., n. sp. a nematode parasite of roots. Proceedings of the Helminthological Society of Washington, D. C. 7:35-42.

1.26. Linford, M. B., and Francis Yap. 1940. Some host plants of the reniform nematode in Hawaii. Proceedings of the Helminthological Society of Washington, D. C. 7:42-44.

1.27. Metha, N., K. K. Wallia, and D. C. Gupta. 1990. Effect of culture filtrates of Rhizoctonia solani and Rhizoctonia bataticola cultured on different media on hatching of Meloidogyne javanica larvae. Plant Disease Research 5:96-99.

7

1.28. Nath, R., M. N. Khan, R. S. Kamalwanschi, and R. P. Dwivedi. 1985. Influence of root knot nematode Meloidogyne incognita on pre and postemergence damping off of tomato. Indian Journal of Nematology 14:135-140.

1.29. Nava, C. 1970. Influence of Meloidogyne incognita on root rot development by Rhizoctonia solani and Pythium ultimum in tomato. M. S. Thesis. N. C. State University, Raleigh, NC.

1.30. Polychronopoulous, A. G., B. R. Houston, and B. T. Lownberry. 1969. Penetration and development of Rhizoctonia solani in sugarbeet seedlings infected with Heterodera schachtii. Phytopathology 59:482-485.

1.31. Powell, N. T., and C. K. Batten. 1967. The influence of Meloidogyne incognita on Rhizoctonia root rot in tobacco. Phytopathology 57:826 (Abstr.).

1.32. Powell, N. T., and C. J. Nusbacum. 1960. The black shank root knot complex in flue cured tobacco. Phytopathology 50:899-906.

1.33. Powell, N. T. 1971. Interaction between nematodes and fungi in disease complexes. Annual Review of Phytopathology 9:253-273.

1.34. Rambir Singh, D. C. Gupta, and K. K. Wallia. 1986. Effect of Rhizoctonia solani culture filtrate on hatching of Meloidogyne javanica larvae. Indian Phytopathology 39:624-625.

1.35. Ramnath and R. D. Dwivedi. 1981. Effect of root knot nematode on development of gram caused by Fusarium oxysporum f. sp. ciceri and root rot by Rhizoctonia sp. Indian Journal of Mycology and Plant Pathology 11:46-49.

1.36. Reddy, P. P., D. B. Singh, and S. R. Sharma. 1979. Interaction of Meloidogyne incognita and Rhizoctonia solani in a root rot disease complex of french bean. Indian Phytopathology 32:651-652.

1.37. Reynolds, H. W., and R. G. Hanson. 1957. Rhizoctonia disease of cotton in presence or absence of the cotton root knot nematode in Arizona. Phytopathology 47:256-261.

1.38. Riedel, R. M. 1988. Interactions of plant-parasitic nematodes with soil-borne plant pathogens. Agriculture, Ecosystems and Environment 24:281-292.

1.39. Schollte, K. S., and J. J. Jacob. 1989. Synergistic interactions between Rhizoctonia solani, Verticillum dahliae, Meloidogyne spp. and Pratylenchus neglectus in potato. Potato Research 32:387-395.

1.40. Siddiqui, M. A., A. Haseeb, and M. M.AIam. 1987. Combined effect of two nematodes and a fungus on the growth and water absorption capability of okra. Indian Journal of Plant Pathology 5:83-86.

1.41. Sikora, R. A., and W. W. Carter. 1987. Nematode interactions with fungal and bacterial plant pathogens, fact or fantasy. Pp. 307-312 in J. A. Veech and D. W. Dickson, eds. Vistas on nematology. Hyattsville, MD: Society of Nematologists.

8

1.42. Sinclair, J. B. 1958. Greenhouse screening of certain fungicides for control of Rhizoctonia damping-off of cotton seedlings. Plant Disease Reporter 42:1084-1088.

1.43. Smith, A. L., and A. L. Taylor. 1941. Nematode distribution in the 1940 regional cotton wilt plots. Phytopathology 31:771 (Abstr.).

1.44. Taylor, C. E. 1990. Nematode interactions with other pathogens. Annals of Applied Biology 116:405-416.

1.45. White, L. V. 1962. Root knot and seedling disease complex of cotton. Plant Disease Reporter 46:501-504.

CHAPTER 2

THE INTERRELATIONSHIPS OF ROTYLENCHULUS RENIFORMIS WITHRHIZOCTONIA SOLANI

9

10

introduction

In the United States, yield loss in cotton (Gossypium spp.) attributed to nematodes is

estimated at 2.4% (2.2). The reniform nematode, Rotylenchulus reniformis Linford and Oliveira,

is recognized as an important pest of cotton in the United States (2.12). It has been reported

from 47 of the 67 parishes in Louisiana and is probably responsible for most of the nematode

damage done to the cotton crop (E. C. McGawley and C. Overstreet, pers. comm.). Seedling

diseases in cotton account for an annual loss of 2.7% in the United States and 2% in Louisiana

(2.2). Rhizoctonia solani Kuhn is the fungus most commonly isolated from diseased cotton

seedlings in Louisiana. Quite often, the population density of R. reniformis in soil is higher in

areas of cotton fields where seedling blight symptoms are apparent, compared with areas in

which seedlings do not exhibit disease symptoms.

Numerous investigators have detailed examples of relationships between Rhizoctonia

solani and nematodes in which their association results in augmented disease incidence and

altered nematode reproduction. Increased incidence of seedling disease caused by R. solani

was noticed in the presence of Meloidogyne spp. on various crops including cotton (2.4-2.6,

2.8, 2.24, 2.25). Resistance to Meloidogyne incognita (Kofoid & White) Chitwood, in pepper

(Capsicum annuum L.) was lost in the presence of R. solani (2.11). R. solani inhibited the

multiplication of M. incognita and R. reniformis in cowpea (Vigna sinensis Endl.) (2.17). In tomato

(Lycopersicon esculentum Mill.), the presence of R. solani enhanced the ratio of males to

females of Heterodera rostochiensis Wollenweber (2.16).

The association of R. reniformis with R. solani in disease complexes in cotton and okra

(Abelmoschus esculentus L.) has been studied by a few researchers (2.1, 2.18, 2.19, 2.26).

Brodie and Cooper (2.1) investigated the involvement of R. reniformis and R. solani in seedling

disease complex of cotton. They found that at 40 nematodes/gram of soil, susceptibility to

postemergence damping-off was greater than that which was observed in the absence of the

nematode. However, their study focused mainly on the severity of seedling blight in the

presence of both pathogens. No information related to the reproduction of R. reniformis was

11

provided. The objectives ot our study were 1) to monitor the influence of R. solani on

reproduction of R. reniformis, 2) to study the impact of R. reniformis on the severity of

Rhizoctonia seedling blight of cotton, and 3) to evaluate the effect of infestation with both

pathogens on cotton growth.

Materials and Methods

Single egg masses of R. reniformis originally isolated from cotton roots collected from

Morehouse and Avoyelles Parishes, Louisiana were increased and maintained on Rutgers

tomato in a greenhouse and are referred to as populations 1 and 2, respectively. Both

populations were confirmed as R. reniformis (2.9) and typed as race A (2.10). Vermiform stages

(juveniles, females and preadult females) of R. reniformis used for inoculum were recovered

from soil by a modified centrifugal-sugar flotation technique (2.14) with nested 425-pm-pore (40

mesh) and 45-pm-pore (325 mesh) sieves. Five-day-old cotton seedlings were transplanted

singly to the center of 15 (experiments 1, 5, and 6) or 10-cm-d (experiments 2, 3, and 4) clay

pots that contained 1000 or 500 g, respectively, of a 3:2:1 mixture of methyl bromide-treated

loamy soil (80.8% sand, 4.7% silt, 14.5% clay), autoclaved sand, and Weblite (Weblite Corp.,

Roanoke, VA). Soil pH was adjusted to 6.5 by adding aluminum sulfate (4 g/Kg of soil). Fifteen-

day-old seedlings were inoculated by pipetting suspensions containing the desired numbers of

nematodes into two depressions (1.0 cm wide x 4.0 cm deep) 2.5 cm from each seedling. At

harvest, plant stems were excised 2.5 cm above the soil surface and fresh weights of shoots

recorded. Root systems were separated from soil by gently agitating the root ball in a 6-L

graduated pitcher containing 4 L water. An additional liter of water was poured over the root

system as it was removed from the pitcher. The soil-water suspension was stirred and

nematodes present in 500 ml of the suspension were extracted (2.14), counted, and total

numbers per pot and rates of reproduction, R (where R = Pf/Pi and Pf equals final population

density and Pi equals inoculum level), were computed (2.7). Each root system was blotted with

paper towels and the weight recorded. Eggs were extracted from root pieces that were

randomly selected and weighed to give a 3.0-g subsample of root system (2.13). Numbers of

12

sessile females were counted on 20 (experiment 1), or 10 (experiments 3, 4 ,5 , and 6) randomly

selected 2.5 cm root segments.

Three isolates (1, 2, and 3) of R. solani were obtained from cotton seedlings exhibiting

seedling blight symptoms in fields in Richland, Rapides and Franklin Parishes, respectively.

Hypocotyl regions were cut into 2-cm sections, disinfested for 3 minutes in 0.5% NaOCI, rinsed

in sterile water, and incubated on 2% water agar at ambient temperature (22-26 C) for 24 hr.

Hyphal tips were transferred to potato dextrose agar for maintenance. The three isolates were

typed as members of Anastomosis Group 4 (2.23). Whole grain oats were soaked overnight in

water (1.5 ml of water/g of dry grain) in wide mouth Mason jars, autoclaved (120 C at 1.09 Kg-

force/cm2) for 45 minutes on 2 successive days, and inoculated with 2 potato dextrose agar

discs (1 cm-d) cut from the growing edge of a 3-day-old fungal culture. After 20 days, the

contents of the jars were removed, mixed, air dried for a week, and stored in plastic bags at 5 C

until use (2.3). Preliminary studies conducted using 1, 2, or 3 oat grains colonized by R. solani

per pot as inoculum showed that a single infested oat grain would elicit a sub-lethal level of

infection. A depression (1.5-cm-d, 1.0 cm deep) was made in soil at a distance of 2.5 cm from

the 15-day-old cotton seedling and a single infested oat grain was added and covered with soil

(experiments 2 ,3 ,4 , 5, and 6). At harvest, disease severity was indexed on a 0-3 scale where 0

= no hypocotyl necrosis or root discoloration; 1 = hypocotyl necrosis, slight root discoloration; 2

= hypocotyl necrosis, moderate root discoloration; and 3 = hypocotyl necrosis, severe root

discoloration. The hypocotyl regions were cut into 2-cm sections, disinfested for 3 minutes in

0.5% NaOCI, rinsed in sterile water and incubated on 2% water agar at ambient temperature for

48 hr to verify the presence of the fungus.

Cotton (G. hirsutum) cultivars Deltapine 90 (DP 90), Deltapine 41 (DP 41) and Stoneville

825 (STV 825), all widely cultivated in Louisiana, were used in the studies. All tests were

conducted in a greenhouse with temperatures ranging from 22-35 C. Plants were fertilized

every two weeks, commencing at 5 days after transplanting, with a 23-19-17 (nitrogen:

phosphorus:potassium) fertilizer solution (800 ppm N, 700 ppm P, and 450 ppm K), and insect

13

and mite populations were controlled as necessary. Supplementary light from fluorescent and

incandescent sources (ca. 260 pE. m-2. s-1. at bench surface) was provided (experiment 6) to

give a 14:10 hr light:dark period each day. The experimental design for all the tests was a

randomized complete block with factorial treatment arrangement. Data were analyzed using the

SAS General Linear Model procedure (2.28). When the number of treatment levels exceeded

two, single-degree-of-freedom contrasts were used to test for differences between levels.

Experiment 1: The objective of this test was to compare the reproduction of R.

reniformis on three cotton cultivars. In this experiment multiple inoculum levels of population 1

of R. reniformis were used. Treatments consisted of four infestation levels of the nematode (0,

500, 2,000, and 8,000/pot; 80% juveniles, 15% males and 5% preadult females) and three

cotton cultivars (DP 90, DP 41, and STV 825), a total of 12 treatment combinations, each

replicated five times. The experiment was terminated after 90 days. Results of this experiment

were used to identify cultivars for more extensive study.

Experiment 2: This experiment examined effects of R. solani isolates 1, 2, and 3 on

population 1 of R. reniformis. Treatments consisted of two nematode infestation levels (0 and

4,000/pot; 87% juveniles, 9% males, and 4% preadult females), four fungal inocula (autoclaved

oat grain, oat grain colonized by R. solani isolate 1, 2, or 3) and one cultivar (DP 90). There were

a total of eight treatment combinations, each replicated five times. The experiment was

terminated after 40 days.

Experiment 3: In this experiment individual and interactive effects of populations 1 and

2, of R. reniformis and R. solani isolates 1 and 2 were studied on two cotton cultivars.

Treatments consisted of two infestation levels of the nematode (0 and 4,000/pot each of the

population 1 or 2; 83% juveniles, 11% males, and 6% preadult females of population 1; 81%

juveniles, 13% males, and 6% preadult females of population 2), three fungal inocula

(autoclaved oat grain, oat grain colonized by R. solani isolate 1 or 2) and two cultivars (DP 90 and

DP 41), a total of 18 treatment combinations, each replicated five times. The experiment was


14

Experiment 4; This study examined the individual and interactive effects of multiple

inoculum levels of nematode population 1 and fungus isolates 1 and 2 on DP 90 and DP 41

cotton. Treatments consisted of three infestation levels of the nematode (0, 500, and

4,000/pot; 83% juveniles, 12% males, and 5% preadult females), three fungal inocula

(autoclaved oat grain, oat grain colonized by R. solani isolate 1 or 2) and two cultivars. There

were 18 treatment combinations, each replicated five times. The experiment was terminated

after 60 days.

Experiment 5; In this study the individual and interactive effects of multiple inoculum

levels of nematode population 1 and fungus isolate 1 were examined. The duration of this

experiment was 90 days. Treatments consisted of four infestation levels of the nematode (0,

500, 2,000, and 8,000/pot; 90% juveniles, 7% males, and 3% preadult females), two fungal

inocula (autoclaved oat grain or oat grain colonized by isolate 1 of R. solani) and the cultivar DP

90, for a total of eight treatment combinations, each replicated five times.

Experiment 6; This study examined the individual and interactive effects of multiple

inoculum levels of R. reniformis population 1 and R. solani isolates 1 and 2 on two cotton

cultivars. Treatments consisted of three infestation levels of the nematode (0, 500, and

4,000/pot; 82% juveniles, 11% males, and 7% preadult females), three fungal inocula

(autoclaved oat grain, oat grain colonized by R. solani isolate 1 or 2) and the cultivars DP 90 and

DP 41. Each treatment combination was replicated five times and the experiment was


Results

Experiment 1: Increasing inoculum levels of R. reniformis resulted in stepwise increases

(P < 0.01) in total numbers of nematodes recovered from soil, sessile females, and eggs/g of

root (Table 2.1). The relationship between nematode inoculum level and reproduction was

linear (P < 0.0001). However, Pf/Pi ratios were inversely related to nematode inoculum level.

Influences of cultivar on nematodes in soil, Pf/Pi ratio, and egg production were significant (P <

0.05). Nematode population densities in soil, Pf/Pi ratios, and eggs/g root were greatest on DP

15

Table 2.1. Population density of Rotylenchulus reniformis on three cotton cultivars at 90 days after inoculation.

Treatment LevelNematodes

in soil Pf/Pi*Sessilefemales§

Eggs/grod

Nematode 500t 24,645 49.3 1 3652,000 73,154 36.6 4 1,8198,000

Contrast144,695 18.1 8 2,212

Linear *** *** *** ***

Cultivar DP 901J 89,513 42.4 6 1,982DP 41 73,939 24.4 3 964STV 825

Contrast79,042 37.1 5 1,450

DP 90 vs DP 41 ** *** * ***

DP 90 vs STV 825 Source

NS * * *

Nematode *** *** ** ***

Cultivar * *** NS ***

N xC * *** NS **

t Vermiform stages per pot (15- cm-d). f DP 90 = Deltapine 90, DP 41 = Deltapine 41, STV 825 = Stoneville 825. t Pi = initial nematode infestation level. Pf = final nematode population density in soil. § Females/20 root segments, each 2.5 cm long. ** * * * * = significant at P = 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

90 followed by STV 825 and DP 41. Nematode x cultivar interaction affected the numbers of

nematodes recovered from soil, Pf/Pi ratios, and eggs/g of root (P < 0.05). Examination of

individual treatment means for nematodes in soil showed that, at lower nematode inoculum

densities (500 and 2,000/pot), numbers of nematodes in soil were the greatest on DP 90

followed by STV 825 and DP 41. However, when the inoculum level was raised to 8,000

nematodes per pot, the greatest numbers of nematodes in soil were recorded for DP 41

followed by DP 90 and STV 825. The same trend was observed for Pf/Pi ratios. On DP 41, as

the inoculum level was raised from 500 to 2,000 nematodes/pot, egg production increased by

360% compared with only a 15% increase when the inoculum level was increased from 2,000 to

8,000. On STV 825 and DP 41 the numbers of eggs/g of root increased in proportion to

inoculum level. Based on these results, DP 90 and DP 41 were chosen for use in subsequent

studies since they supported higher and lower nematode populations, respectively. Increasing

inoculum levels of R. reniformis resulted in stepwise reductions (P < 0.05) in shoot, root, and

plant fresh weights (Table 2.2). Shoot, root and plant fresh weights were the highest for DP 41

and lowest for DP 90.

Experiment 2 : Colonization of cotton seedlings by R. solani resulted in an increase (P <

0.01) in juveniles and total nematodes recovered from soil, Pf/Pi ratios, and eggs/g of root

(Table 2.3). There were differences (P < 0.05) between R. solani isolates 1 and 2 with respect to

their impact on juveniles and total nematodes recovered from soil and Pf/Pi ratios. Fungus

isolates 2 and 3 differed regarding their influence on egg production (P < 0.0001). At 40 days

after inoculation, R. reniformis did not influence either plant growth parameters or disease

indices (Table 2.4). The fungus reduced (P < 0.0001) shoot, root, and plant fresh weights. Each

isolate had an effect on plant growth but there were no differences between isolates.

Experiment 3 : The only observed difference in reproduction (P < 0.05) between the

two reniform nematode populations was in the numbers of juveniles recovered from soil (Table

2.5). The presence of R. solani resulted in an increase (P < 0.05) in juveniles, males, and total

17

Table 2.2. Effect of Rotylenchulus reniformis on fresh weights of three cotton cultivars at 90 days after inoculation.

Treatment Level

Plant fresh weight (grams)

Shoot Root Plant

Nematode 0 59.0 41.4 100.4500t 56.2 41.1 97.3

2,000 55.3 40.0 95.3

Contrast8,000 54.8 38.1 92.9

Linear * f t * f t *

Cultivar DP 90H 49.6 34.6 84.1DP 41 67.2 46.8 114.0

ContrastSTV 825 52.2 39.2 91.4

DP 90 vs DP 41 f t * * * * * * * *

DP 90 vs STV 825 Source

* * * * ft**

Nematode ** * ft*

Cultivar * * * ftftft * * *

N xC NS NS NS

t Nematode levels correspond to vermiform stages per pot (15-cm-d). K DP 90 = Deltapine 90, DP 41 = Deltapine 41, STV 825 = Stoneville 825. *,**,*“ = significant at P = 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

18

Table 2.3. Effects of Rhizoctonia solani isolates on Rotylenchulus reniformis populations on Deltapine 90 cotton at 40 days after inoculation.

Treatment Level

Life stages in soil

Juveniles Males Females Total Pf/P itEggs/groot

Fungus None 2,963 211 73 3,247 0.8 1,342Isolate 1 4,100 264 107 4,471 1.1 2,373Isolate 2 3,358 231 99 3,688 0.9 2,332Isolate 3 3,148 297 99 3,544 0.9 1,569

Contrast0 vs1+2+3 * NS NS * * * * *

1 V S 2 * NS NS * * NS2 vs 3 NS NS NS NS NS * * *

SourceFungus ** NS NS ** * * * * *

t Pi = initial nematode infestation level (4,000/pot of 10-cm-d). Pf = final nematode population density in soil. W * * = significant at P = 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

19

Table 2.4. Effects of Rotylenchulus reniformis and Rhizoctonia solani isolates on fresh weights and disease indices of Deltapine 90 cotton at 40 days after inoculation.

Treatment Level


Shoot Root PlantDiseaseindex$

Nematode 0 4.8 4.0 8.8 1.34,000f 4.8 4.2 9.0 1.3

Fungus None 6.1 5.1 11.2 0.0Isolate 1 4.1 3.8 7.9 1.8Isolate 2 4.4 3.7 8.1 1.9Isolate 3 4.5 3.7 8.2 1.8

ContrastOvs 1+2+3 *** *** *** ***

1 vs 2 NS NS NS NS2 vs 3 NS NS NS NS

SourceNematode NS NS NS NSFungus *** *** *** ***

N xF NS NS NS NS

t Vermiform stages per pot (10-cm-d). t Disease index scale = 0-3 (0 = no hypocotyl necrosis or root discoloration; 1 = hypocotyl necrosis, slight root discoloration; 2 = hypocotyl necrosis, moderate root discoloration; 3 = hypocotyl necrosis, severe root discoloration) *,“ ,*** = significant at P = 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

Table 2.5. Effects of Rhizoctonia solani isolates and cotton cultivars on density of two populations of Rotylenchulus reniformis at 40 days afterinoculation.

Treatment Level

Life stages in soil

Juveniles Males Females Total Pf/Pi*Sessile

females§Eggs/groot

Nematode Population 1 f 1,723 150 37 1,909 0.5 1.4 423Population 2 1,848 139 34 2,021 0.5 1.8 442

Fungus None 1,261 122 23 1,406 0.4 0.8 351Isolate 1 2,140 158 42 2,339 0.6 2.0 486Isolate 2 2,015 156 43 2,213 0.6 2.1 470

ContrastOvs 1+2 * * * * * * * * * * * * * * * *

1 vs 2 NS NS NS NS NS NS NS

Cultivar DP 90fl 2,045 151 42 2,238 0.6 1.9 535DP 41 1,526 138 28 1,692 0.4 1.3 330

SourceNematode * NS NS NS NS NS NSFungus * * * * NS * * * * * * * * * * ★

Cultivar * * * NS NS * * * * * * * * * *

N xF NS NS NS NS NS NS *

N xC NS NS NS NS NS NS NSFxC * * NS NS * * * * NS *

N x F x C NS NS NS NS NS NS NS

t Vermiform stages of 4,000 nematodes per pot (10-cm-d). DP 90 = Deltapine 90, DP 41 = Deltapine 41. $ Pi = initial nematode infestation level. Pf = final nematode population density in soil. § Females/10 root segments, each 2.5 cm long. *,**,*** = significant at P = 0.05, 0.01 and 0.0001 based on Ftest, respectively. NS = nonsignificant.

too

nematodes. Pf/Pi ratios, numbers of sessile females, and eggs/g of root were also greater in the

presence of R. solani (P < 0.01). The two Rhizoctonia isolates did not differ in their influence on

reproduction by R. reniformis. Juveniles, total nematodes from soil, Pf/Pi ratios, numbers of

sessile females, and eggs/g of root were greater (P < 0.05) on DP 90 than on DP 41. The

numbers of eggs/g of root were affected by interactions between fungus and nematode (P <

0.05). Fungus isolate 1 had a more pronounced influence on egg production by population 1

than did isolate 2. In contrast, R. solani isolate 2 had a greater influence on egg production by

population 2 than did isolate 1. Fungus x cultivar interaction influenced (P < 0.05) the numbers

of juveniles and total nematodes recovered from soil, Pf/Pi ratios, and eggs/g of root. Treatment

mean patterns for juveniles (Fig. 2.1) revealed that DP 90 which supports higher levels of R.

reniformis, allowed a greater increase in soil juveniles in response to R. solani than did DP 41,

which supports lower levels of R. reniformis. Similar trends were obtained for total nematodes

present in soil, Pf/Pi ratios, and eggs/g of root. Neither plant growth nor disease indices were

significantly influenced by R. reniformis in this 40-day-duration experiment (Table 2.6). The

fungus caused reductions (P < 0.0001) in shoot, root, and plant fresh weights, but there were

no differences between isolates. Shoot and plant fresh weights for DP 90 were lower than

those for DP 41 (P < 0.0001). Fungus x cultivar interaction affected fresh shoot and plant

weights (P < 0.0001). Examination of treatment means revealed that reductions in shoot and

plant weights caused by both fungus isolates were greater on DP 41 than on DP 90.

Experiment 4 : Increasing the inoculum level of R. reniformis from 500 to 4,000

nematodes per pot resulted in an increase (P < 0.01) in all the life stages in both soil and roots

(Table 2.7). Pf/Pi ratios were inversely related to nematode inoculum levels. Parasitism of cotton

seedlings by R. solani resulted in an increase (P < 0.01) in all the nematode developmental

stages except sessile females. No differences were observed between R. solani isolates in their

impact on nematode reproduction. Life stages of the nematode recovered from soil, Pf/Pi ratio,

and eggs/g of root were greater (P < 0.01) on DP 90 than on DP 41. Pf/Pi ratio was affected by

nematode x fungus interaction (P < 0.0001). Inspection of individual treatment mean patterns

Juve

nile

s in

soil

3000

2500-

2000 -

1500-

1000 -

500-

None *lsol. 1 Isol. 2 None Isol. 1 Isol. 2

DP 90 DP 41

Fungus/Cultivar levels

Fig. 2.1. Means for the juveniles of Rotylenchulus reniformis recovered from soil in experiment 3, for the interaction between Rhizoctonia solani and cotton cultivar. Vertical lines delimit standard errors of means. *lsol. 1 = Isolate 1, Isol. 2 = Isolate 2.

PON>

23

Table 2.6. Effects of Rotylenchulus reniformis populations, Rhizoctonia solani isolates and cultivars on cotton fresh weights and disease indices at 40 days after inoculation.

Treatment Level


Shoot Root PlantDiseaseindex}:

Nematode None 4.4 3.1 7.5 1.2Population 1 f 4.3 3.2 7.5 1.3Population 2 4.2 3.2 7.4 1.2

Contrast0 vs 1+2 NS NS NS NS1 VS2 NS NS NS NS

Fungus None 5.6 3.8 9.4 0.0Isolate 1 3.7 2.9 6.5 1.9Isolate 2 3.6 2.7 6.3 1.9

ContrastOvs 1+2 * 4 * * 4 4 * 4 * 444

1 vs 2 NS NS NS NS

Cultivar DP 90K 3.7 3.1 6.8 1.3DP 41 5.0 3.2 8.2 1.2

SourceNematode NS NS NS NSFungus * 4 * * 4 4 4 4 * * 4 4

Cultivar 4 4 * NS * 4 * NSN xF NS NS NS NSN xC NS NS NS NSFxC 4 4 * NS 444 NSN x F x C NS NS NS NS

t Vermiform stages of 4,000 nematodes per pot (10-cm-d). H DP 90 = Deltapine 90, DP 41 = Deltapine 41. 4: Disease index scale = 0-3 (0 = no hypocotyl necrosis or root discoloration; 1 = hypocotyl necrosis, slight root discoloration; 2 = hypocotyl necrosis, moderate root discoloration; 3 = hypocotyl necrosis, severe root discoloration).*,**,*** = significant at P= 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

Table 2.7. Effects of nematode inoculum levels, Rhizoctonia solani isolates and cotton cultivars on population density of Rotylenchulus reniformisat 60 days after inoculation.

Treatment Level Juveniles

Life stages in soil

Males Females Total Pf/Pi*Sessile

females§Eggs/g

root

Nematode 500f 1,770 120 25 1,914 3.8 0.5 2344,000 2,909 202 61 3,172 0.8 1.9 500

Fungus None 1,881 124 26 2,031 1.8 0.9 312Isolate 1 2,581 178 49 2,808 2.7 1.4 392

Contrastisolate 2 2,626 186 55 2,868 2.4 1.4 409

0 vs 1+2 444 444 44 444 444 NS 444

1 vs2 NS NS NS NS NS NS NS

Cultivar DP 90fl 2,690 184 53 2,927 2.6 1.4 456

SourceDP 41 1,997 138 32 2,168 1.9 1.0 280

Nematode 444 444 444 444 * * * 44 444

Fungus * * * 444 44 444 444 NS 44

Cultivar * * * 44 44 444 444 NS 444

N xF NS NS NS NS 444 NS NSNxC NS NS * NS 444 NS NSFxC NS NS NS NS NS NS NSN x F x C NS NS NS NS NS NS NS

t Vermiform stages per pot (10-cm-d). % DP 90 = Deltapine 90, DP 41 = Deltapine 41. $ Pi = initial nematode infestation level. Pf = final nematode population density in soil. § Females/10 root segments, each 2.5 cm long. *,**,*“ = significant at P = 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

ro

revealed an inverse relationship between inoculum levels of the nematode and Pf/Pi ratios

either in the absence or presence of the fungus. Nematode x cultivar interaction impacted (P <

0.05) the numbers of females in soil as well as Pf/Pi ratio. At the 500 nematodes per pot

inoculum level, numbers of females in soil were similar on both cultivars. However, when the

inoculum level was increased to 4,000 per pot there was a 200% increase in the numbers of

females on DP 90, compared with a 78% increase on DP 41. Inspection of individual treatment

mean patterns for Pf/Pi ratio revealed an inverse relationship between inoculum levels and Pf/Pi

ratios on DP 90 as well as on DP 41. Moreover, the magnitude of difference in Pf/Pi ratio

between DP 90 and DP 41 was greater at the 500 than at the 4,000 nematodes/pot inoculum

level. The effect of the nematode on plant parameters and disease indices was not significant at

60 days after inoculation (Table 2.8). R. solani caused reductions (P < 0.0001) in shoot, root,

and plant fresh weights. However, there were no differences between the two isolates in their

influence on plant growth. Root and plant weights for DP 90 were lower than those for DP 41 (P

< 0.01).

Experiment 5 : Increasing inoculum levels of R. reniformis resulted in stepwise increases

(P < 0.05) in nematode life stages recovered from the soil, sessile females associated with roots,

and eggs/g of root (Table 2.9). A linear relationship existed between nematode inoculum levels

and reproduction (P < 0.0001). Pf/Pi ratios were inversely related to inoculum levels. The

presence of R. solani in cotton increased (P < 0.05) the numbers of juveniles, males, and total

nematodes recovered from soil. Pf/Pi ratio as well as egg production was greater in the

presence of the fungus (P < 0.01). Juveniles and total nematodes in soil were affected by

nematode x fungus interaction (P < 0.05). The presence of R. solani at the 8,000

nematodes/pot inoculum level resulted in greater increase in juveniles than the 500 or 2,000

per pot levels. A similar trend was observed for total nematodes in soil. At 90 days after

inoculation there were stepwise reductions (P < 0.0001) in fresh shoot, root, and plant weights

which parallelled increases in nematode inoculum levels (Table 2.10). R. reniformis did not

influence the disease severity. R. solani caused reductions (P < 0.0001) in plant growth. The

26

Table 2.8. Effects of inoculum levels of Rotylenchulus reniformis and Rhizoctonia solani isolates on fresh weights and disease indices of two cotton cultivars at 60 days after inoculation.

Treatment Level

Plant fresh weights (grams)

Shoot Root PlantDiseaseindext

Nematode 0 9.9 9.2 19.2 1.1500t 10.0 9.1 19.1 1.3

Contrast4,000 9.8 9.0 18.9 1.3

Linear NS NS NS NSQuadratic NS NS NS NS

Fungus None 11.6 10.9 22.5 0.0Isolate 1 9.0 8.2 17.1 1.9

ContrastIsolate 2 9.0 8.1 17.1 2.0

0 vs 1+2 * * * * * * * * * * * *

1 vs 2 NS NS NS NS

Cultivar DP 90U 9.8 8.8 18.6 1.3

SourceDP 41 10.0 9.5 19.5 1.2

Nematode NS NS NS NSFungus * * * * * * * * * ***

Cultivar NS * * * * * NSN xF NS NS NS NSN xC NS NS NS NSFxC NS NS NS NSN x F x C NS NS NS NS

t Vermiform stages per pot (10-cm-d). H DP 90 = Deltapine 90, DP 41 = Deltapine 41. t Disease index scale = 0-3 (0 = no hypocotyl necrosis or root discoloration; 1 = hypocotyl necrosis, slight root discoloration; 2 = hypocotyl necrosis, moderate root discoloration; 3 = hypocotyl necrosis, severe root discoloration). ♦,**,*** = significant at P = 0.05, 0.01 and 0.0001 based on Ftest, respectively. NS = nonsignificant.

Table 2.9. Effects of nematode inoculum levels and Rhizoctonia solani on population density of Rotylenchulus reniformis on Deltapine 90 cotton at90 days after inoculation.

Treatment Level

Life stages in soil

Juveniles Males Females Total Pf/Pi*Sessile

females§Eggs/g

root

Nematode 500f 46,420 3,593 953 50,967 101.9 0.7 9802,000 117,700 10,560 1,687 129,947 64.9 2,0 2,2498,000 215,952 18,282 2,904 237,138 29.6 2.6 3,088

ContrastLinear *** *** *** 1rtrk *** ♦ irk*

Fungus None 116,248 9,504 1,584 127,336 60.2 1.4 1,933Isolate 1 145,606 12,895 2,234 160,735 68.9 2.2 2,380

SourceNematode *** *** irk ■kirk *** * ***

Fungus irk * NS ■kirk ** NS ***

N xF * NS NS * NS NS NS

t Vermiform stages per pot (15-cm-d) respectively. Pi = initial nematode infestation level. Pf = final nematode population density in soil.§ Females/10 root segments, each 2.5 cm long. *,**,*“ = significant at P = 0.05,0.01 and 0.0001 based on Ftest, respectively.NS = nonsignificant.

ro-v |

28

Table 2.10. Effects of inoculum levels of Rotylenchulus reniformis and Rhizoctonia solani on fresh weights and disease indices of Deltapine 90 cotton at 90 days after inoculation.

Treatment Level


Shoot Root PlantDiseaseindext

Nematode 0 25.1 17.0 42.1 0.8500t 22.6 15.8 38.4 0.7

2,000 21.5 14.8 36.4 0.78,000 20.3 13.7 34.0 0.6

ContrastLinear * * * *** *** NS


SourceNematode * * * *** * * * NSFungus *** * * * ■kit it ***

N xF * * * * * * NS

t Vermiform stages per pot (15-cm-d). % Disease index scale = 0-3 (0 = no hypocotyl necrosis or root discoloration; 1 = hypocotyl necrosis, slight root discoloration; 2 = hypocotyl necrosis, moderate root discoloration; 3 = hypocotyl necrosis, severe root discoloration). * * * * * * = significant at P = 0.05, 0.01 and 0.0001 based on Ftest, respectively. NS = nonsignificant.

29

interaction between nematode x fungus influenced shoot, root weights (P < 0.01) and effects

on plant growth were antagonistic (Fig. 2.2). That is, reductions in plant growth caused by both

pathogens together was less than the sum of reductions caused by each alone.

Experiment 6 : increasing the inoculum level of R. reniformis from 500 to 4,000

nematodes per pot resulted in an increase (P < 0.05) in all the life stages of the nematode in soil

and egg production on roots (Table 2.11). Pf/Pi ratios were inversely related to nematode

inoculum levels. Presence of R. solani resulted in an increase (P < 0.05) in all the life stages of

the nematode except sessile females. The two isolates of the fungus did not differ in their

influence on reproduction of R. reniformis. Juveniles, total nematodes from soil, Pf/Pi ratio and

eggs/g of root were greater (P < 0.01 ) on DP 90 than on DP 41. Pf/Pi ratio was affected by

interactions between fungus and nematode (P < 0.05). Examination of individual treatment

means revealed an inverse relationship between inoculum levels of the nematode and Pf/Pi

ratios either in the absence or presence of the fungus. The interaction between nematode and

cultivar influenced (P < 0.01) Pf/Pi ratio and inspection of individual treatment means revealed

an inverse relationship between nematode inoculum levels and Pf/Pi ratios on DP 41 and DP

90. The magnitude of difference in Pf/Pi ratio between DP 90 and DP 41 was greater at 500

than at 4,000 nematodes/pot. R. reniformis caused reductions in all plant growth parameters (P

< 0.0001) (Table 2.12). R. solani reduced shoot, root, and plant fresh weights ( P < 0.0001).

Shoot, root, and plant fresh weights were higher for DP 41 than for DP 90. The interaction

between nematode and fungus was nonsignificant and effects on plant growth were additive.

Discussion

Three general conclusions can be made on the basis of data presented herein. 1) The

presence of R. solani augments reproduction by R. reniformis. 2) R. reniformis has no

detectable influence on severity of cotton seedling blight of cotton. 3) Plant growth effects

caused by the nematode and the fungus together are antagonistic.

Plan

t fre

sh

weig

ht (

g)

5 0 -

4 0 -

3 0 -

20 -

1 0 -

*-F +F +FF +FF +F .cI

**0 500 2,000 8,000

Fungus/Nematode levels

Fig. 2.2. Means for plant fresh weights for the interaction between Rhiozoctonia solani and Rotylenchulus reniformis in experiment 5. Vertical lines delimit standard errors of means. *-F = absence of fungus, +F = presence of fungus. **0, 500, 2,000, and 8,000 nematodes per pot respectively.

uo

Table 2.11. Effects of nematode inoculum levels, Rhizoctonia solani isolates and cotton cultivars on population density of Rotylenchulus reniformisat 90 days after inoculation.

Treatment Level Juveniles

Life stages in soil

Males Females Total Pf/Pi*Sessile

females§Eggs/g

root

Nematode 500f 30,525 1,936 583 33,044 66.1 1.1 7874,000 54,905 2,435 819 58,160 14.5 2.0 1,406

Fungus None 37,290 1,782 545 39,617 34.2 1.2 887Isolate 1 43,838 2,362 868 47,068 43.9 1.6 1,140

Contrastisolate 2 46,464 2,409 693 49,566 44.3 1.7 1,250

0 vs 1+2 * * * * * * * * * * * NS * * *

1 vs 2 NS NS NS NS NS NS NS

Cultivar DP 90% 44,880 2,253 751 47,884 45.2 1.7 1,281

SourceDP 41 40,216 2,112 649 42,977 36.4 1.4 909

Nematode * * * * * * * * * * ★ * NS * * *

Fungus * * * * * * * * * * NS * * *

Cultivar * * NS NS * * * * NS * * *

N xF NS NS NS NS * NS NSN xC NS NS NS NS * * NS NSFxC NS NS NS NS NS NS NSN x F x C NS NS NS NS NS NS NS

t Vermiform stages per pot (15-cm-d). ^ DP 90 = Deltapine 90, DP 41 = Deltapine 41. * Pi = initial nematode infestation level. Pf = final nematode population density in soil. § Females/10 root segments, each 2.5 cm long. *,**,*“ = significant at P = 0.05, 0.01, and 0.0001 based on Ftest, respectively. NS = nonsignificant.

»

32

Table 2.12. Effects of inoculum levels of Rotylenchulus reniformis, Rhizoctonia solani isolates and cotton cultivars on fresh weights and disease indices at 90 days after inoculation.

Treatment Level


Shoot Root PlantDiseaseindex£

Nematode 0 27.8 20.9 48.6 0.9500f 25.5 19.5 45.0 0.9

Contrast4,000 24.4 18.5 42.9 1.0

Linear * * * * * * * * * NS


ContrastIsolate 2 23.7 17.7 41.4 1.4

0 vs 1+2 * * ★ * * * * * * * * *

1 vs 2 NS NS NS NS

Cultivar DP 901 20.5 14.5 35.0 1.0

SourceDP 41 31.3 24.8 56.1 0.9

Nematode f t # * * * * * * * NSFungus * * * * * * * * *

Cultivar * * * * * * * * * NSN xF NS NS NS NSN xC NS NS NS NSFxC NS NS NS NSN x F x C NS NS NS NS

t Vermiform stages per pot (15-cm-d). 1 DP 90 = Deltapine 90, DP 41 = Deltapine 41. $ Disease index scale = 0-3 (0 = no hypocotyl necrosis or root discoloration; 1 = hypocotyl necrosis, slight root discoloration; 2 = hypocotyl necrosis, moderate root discoloration; 3 = hypocotyl necrosis, severe root discoloration). *,**,*** = significant at P = 0.05,0.01 and 0.0001 based on Ftest, respectively. NS = nonsignificant.

Most investigations (2.1, 2.4, 2.6, 2.8, 2.25, 2.30) of interrelationships between R.

solani and either reniform or root knot nematodes have focused mainly on effects of nematodes

on the incidence or severity of disease. Few reports detail the influence of R. solani on

reproduction by R. reniformis (2.17, 2.18). In our studies, enhanced reproduction by R.

reniformis in the presence of R. solani was detectable within 40 days of inoculation. This

increased reproduction occurred with three isolates of R. solani, two populations and four

inoculum levels of R. reniformis and two cultivars of cotton. Significant increases in populations

of R. reniformis in soil were accounted for by the increased production of eggs. In an attempt to

include all life stages of the nematode in the population census, an effort was made to account

for the numbers of females present on the root systems. In one experiment (3) there was a

significant increase in the numbers of sessile females in the presence of R. solani and in three

experiments (4, 5, and 6) there were no significant increases in numbers of sessile females. This

is attributed to the fact that the pattern of infection was not uniform across the plant root system.

In view of the nonuniformity of infection, it was not possible to accurately account for the

numbers of females present on cotton plants which had progressed past the seedling stage.

The significant increase in total nematode population density in soil in the presence of R. solani

resulted from the numbers of juveniles rather than from numbers of males or preadult females.

R. solani attacks the hypocotyl region of the cotton seedling near the soil line and causes

postemergence damping-off which is often called soreshin disease (2.27). During preliminary

studies it was determined that the isolates of R. solani used in our studies do not parasitize the

root system. Reniform nematode remains confined to lateral roots and rarely, if ever, infects the

tap root. Therefore, it is probable that any fungus related effects on nematode reproduction

were indirect via alterations in host physiology. Obviously, this fungus does not have the same

stimulatory effect on all reniform nematode populations, races or hosts since the work of Kumar

and Sivakumar (2.18) with R. solani and R. reniformis in okra indicated that there was no fungus

influence on nematode reproduction.

34

In experiment 3, reproduction of both populations of R. reniformis was enhanced in the

presence of R. solani. This uniformity of influence apparently does not apply across populations

of nematode species since Olthof (2.20) reported that in tobacco Thielaviopsis basicola (Berk

and Br.) Ferraris promoted the development of one population of Pratylenchus penetrans

Cobb, but had no effect on another. In experiments 3 ,4 , and 6 both the isolates of R. solani did

not differ in their influence on reproduction of R. reniformis. Conversely, however, in tomato

Overman and Jones (2.21) observed a 13-fold increase in the population of Tylenchorhynchus

capitatus in the presence of one isolate of Verticillium-albo-atrum Reinke and Berth compared

with a 9-fold increase with another isolate of the fungus.

Experiments 3, 4, and 6, which were of 40, 60, and 90-day-durations, revealed the

same relationship with respect to the nematode host status of the two cotton cultivars. That is, in

either the presence or absence of R. solani, DP 90 was a better host than DP 41 (2.15). This

host status was also consistent in experiment 1, although there was no fungus component

present. Similar observations were made when two pepper cultivars were inoculated with R.

solani and M. incognita (2.11).

In four experiments (1, 4, 5, and 6) in which nematode levels ranged from 0.5-8/g of

soil, Pf/Pi ratios were inversely related to initial inoculum densities. Our observations in both 60

and 90-day-duration experiments (4, 5, and 6) indicated that, at all the nematode inoculum

levels, the presence of R. solani enhanced reproduction of reniform nematode. In soybean

(Glycine max (L.) Merr.), Overstreet et al (2.22) observed that the presence of Calonectria

crotalariae (Loss) Bell and Sobers increased the reproduction of Heterodera glycines Ichinohe

at high, but not low nematode inoculum levels.

Feeding injury caused by the nematode to the cotton root system did not influence the

severity of seedling blight in our experiments in which nematode inoculum levels ranged from

0.5-8/g of soil. Brodie and Cooper (2.1) reported that at inoculum level of 8 nematodes/g of soil,

R. reniformis also had no influence on postemergence damping-off of cotton caused by R.

solani. This fungus, however, when combined with the root knot nematode M. incognita

35

increased the disease severity or incidence of cotton seedling blight (2.4, 2.6, 2.8, 2.30). Also,

in okra the presence of reniform nematode caused wilting symptoms to appear earlier than

those observed with R. solani alone (2.18).

All isolates of R. solani used in our studies were virulent. After many attempts to employ

mycelial mat slurries or infested potato dextrose agar discs as inoculum units to establish

sublethal infections of R. solani, the infested oat grain technique proved to be successful. The

fungus reduced cotton growth as noted in experiments 2-6. The nematode alone caused

significant plant damage at 90 days and combined with the fungus, the relationship was

antagonistic to plant growth. C. crotalariae and H. glycines had a similar antagonistic effect on

soybean growth, even though the presence of the fungus enhanced the nematode

reproduction (2.22). Tchatchoua and Sikora (2.29) found that R. reniformis alone caused

significant reduction in shoot and root weights of cotton at population densities twice that

employed in our studies, and combined inoculations with Verticillium dahliae Kleb. resulted in

synergistic reductions in plant growth. In our research the highest nematode inoculum level was

8/g of soil since this represents the average density of R. reniformis encountered in cotton

fields in Louisiana.

Our research demonstrates that there is an interrelationship between R. solani and

reniform nematode. The fungus, therefore, impacts the cotton plant directly during the pre and

postemergence growth stages and indirectly during later stages through its stimulatory effect on

reproduction of R. reniformis.

Summary

The interrelationships between reniform nematode (Rotylenchulus reniformis) and

cotton (Gossypium hirsutum) seedling blight fungus (Rhizoctonia solani) were studied using

three isolates of R. solani, two populations of R. reniformis at multiple inoculum levels and two

cotton cultivars. Colonization of cotton hypocotyl by R. solani resulted in significant increases in

nematode populations in soil and eggs recovered from the root systems in 40, 60, and 90-day-

duration experiments. Increases in soil populations resulted mainly from increases in juveniles.

Enhanced reproduction of Ft. reniformis in the presence of Ft. solani was consistent across

isolates (1, 2 and 3) of R. solani, populations (1 and 2) and inoculum levels (0.5,1, 2 ,8/g of soil)

of R. reniformis regardless of cotton cultivars (DP 90 and DP 41). Severity of seedling blight was

not influenced by the nematode. R. solani caused significant reductions in cotton growth in 40,

60, and 90 day periods. R. reniformis reduced cotton growth at 90 days. The relationship

between nematode inoculum levels and plant growth reductions was linear. At 90 days when

both the pathogens were together, effects on cotton growth were antagonistic. The presence

of the fungus also impacts cotton growth indirectly by augmenting reproduction and damage

potential of a very pathogenic nematode species.

37

Literature Cited

2.1. Brodie, B. B.f and W. E. Cooper. 1964. Relation of parasitic nematodes to postemergence damping-off of cotton. Phytopathology 54:1023-1027.

2.2. Balsingame, D. 1992. Cotton Disease Loss Estimate Committee report. Pp. 165 in D. J. Herber and D A. Richter, eds. Proceedings of Beltwide Cotton Conferences, vol. Memphis, TN: National Cotton Council.

2.3. Carling, D. E., and D. R. Sumner. 1992. Rhizoctonia. Pp. 157-165 in L. L. Singleton, J. D. Mihail, and C. M. Rush, eds. Methods for Research on Soilborne Phytopathogenic Fungi. St. Paul, MN: APS press.

2.4. Carter, W. W. 1975. Effect of soil temperatures and inoculum levels of Meloidogyne incognita and Rhizoctonia solani on seedling disease of cotton. Journal of Nematology 7:229-233.

2.5. Carter, W. W. 1975. Effect of soil texture on the interaction between Rhizoctonia solani and Meloidogyne incognita on cotton seedlings. Journal of Nematology 7:234-236.

2.6. Carter, W. W. 1981. The effect of Meloidogyne incognita and tissue wounding on severity of seedling disease of cotton caused by Rhizoctonia solani. Journal of Nematology 13:374-376.

2.7. Casewell, E. P., J. DeFrank, W. J. Apt, and C. S. Tang. 1991. Influence of nonhost plants on population decline of Rotylenchulus reniformis. Journal of Nematology 23:91-98.

2.8. Cauquil, J. E., and R. L. Shepherd. 1970. Effect of root knot nematode fungi combinations on cotton seedling disease. Phytopathology 60:448-451.

2.9. Dasgupta, D. R., D. J. Raski, and S. J. Sher. 1968. A revision of the genus Rotylenchulus Linford and Oliveira, 1940 (Nematoda: Tylenchida). Proceedings of the Helminthological Society of Washington 35:169-192.

2.10. Dasgupta, D. R., and A. R. Seshadri. 1971. Races of reniform nematode Rotylenchulus reniformis Linford and Oliveira, 1940. Indian Journal of Nematology 1:21- 24.

2.11. Hasan, A. 1985. Breaking resistance in chilli to root knot nematode by fungal pathogens. Nematologica 31:210-217.

2.12. Heald, C. M., and A. F. Robinson. 1990. Survey of current distribution of Rotylenchulus reniformis in the United States. Supplement to Journal of Nematology 22:695-699.

2.13. Hussy, R. S., and K. R. Barker. 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reporter 57:1025-1028.

2.14. Jenkins, W. R. 1964. A rapid centrifugal flotation technique for separating n e m a t o d e s from soil. Plant Disease Reporter 48:692.

38

2.15. Jones, F. G. W. 1956. Soil population of beet eelworm (Heterodera schachtii Schm.) in relation to cropping. II Microplot and field plot results. Annals of Applied Biology 44:25-56.

2.16. Ketudat, U. 1969. The effect of some soil-borne fungi on the sex ratio of Heterodera rostochiensis on tomato. Nematologica 15:229-233.

2.17. Khan, T. A., and S. I. Husain. 1990. Studies on the effect of interactions of variable inoculum levels of Rotylenchulus reniformis, Meloidogyne incognita and Rhizoctonia solani on cowpea. Current Nematology 1:49-52.

2.18. Kumar, S., and C. V. Sivakumar. 1981. Disease complex involving Rotylenchulus reniformis and Meloidogyne incognita on okra. Nematologia Mediterranea 9:145-149.

2.19. Lambe, R. C., and W. Horne. 1963. The reniform nematode in cotton in the lower Rio Grand valley of Texas. Plant Disease Reporter 47:941.

2.20. Olthof, T. H. A. 1968. Races of Pratylenchus penetrans and their effect on black rot resistance to tobacco. Nematologica 14:482-488.

2.21. Overman, A., and J. P. Jones. 1970. Effect of stunt and root knot nematodes on Verticillium wilt of tomato. Phytopathology 60:1306 (Abstr.)

2.22. Overstreet, C., E. C. McGawley and J. S. Russin. 1990. Interactions between Calonectria crotalariae and Heterodera glycines on soybean. Journal of Nematology 22:496-505.

2.23. Parameter, J. R., R. T. Sherwood, and W. D. Platt. 1969. Anastomosis grouping among isolates of Thanatephorous cucumeris. Phytopathology 59:1270-1278.

2.24. Powell, N. T., and C. K. Batten. 1967. The influence of Meloidogyne incognita on Rhizoctonia root rot in tobacco. Phytopathology 57:826 (Abstr.).

2.25. Reddy, P. P., D. B. Singh, and S. R. Sharma. 1979. Interaction of Meloidogyne incognita and Rhizoctonia solani in a root rot disease complex of french bean. Indian Phytopathology 32:651-652.

2.26. Siddiqui, M. A., A. Haseeb, and M. M.AIam. 1987. Combined effect of two n em ato d es and a fungus on the growth and water absorption capability of okra. Indian Journal of Plant Pathology 5:83-86.

2.27. Sinclair, J. B. 1965. Cotton seedling diseases and their control. Bulletin of Louisiana State University Agricultural Experiment Station 590:1-35.

2.28. SAS Institute Inc. 1989. SAS User’s Guide, Version 6, First Edition, Cary, NC: SAS Institute Inc., 479 pp.

2.29. Tchutchoua, A. and R. A. Sikora. Alterations in susceptibility of wilt resistant cotton varieties to Verticillium dahliae induced by Rotylenchulus reniformis. Zectschrift fur Pflanzenkrankheiten und Pflanzenschutz 90:232-237.

39

2.30. White, L. V. 1962. Root knot and seedling disease complex of cotton. Plant Disease Reporter 46:501-504.

CHAPTER 3

INFLUENCE OF RHIZOCTONIA SOLANI ON EGG HATCHING AND INFECTIVITY OF ROTYLENCHULUS RENIFORMIS

40

41

Introduction

Numerous reports detail fungus-related enhancement of nematode reproduction. For

example, the presence of Verticillium dahliae Kleb. resulted in increased reproduction of the

lesion nematode, Pratylenchus spp. in egg plant {Solatium melongena L.) (3.13), tomato

{Lycopersicon esculentum Mill.) (3.15), and peppermint (Mentha piperita L.) (3.5, 3.6).

Enhanced reproduction of the lesion nematode Pratylenchus penetrans (Cobb) was also

observed to occur in alfalfa {Medicago sativa L.) in the presence of Fusarium oxysporum

Schlecht (3.3, 3.4). Research conducted in Louisiana by McGawley et al. (3.12), Overstreet and

McGawley (3.16), Overstreet et al. (3.17) and, Winchell and McGawley (3.23) have described

interrelationships between nematodes and plant pathogenic fungi in which nematode

reproduction was augmented by the presence of a fungus. Data presented in chapter 2

documented Rhizoctonia solani kuhn related enhancement of reproduction by the reniform

nematode, Rotylenchulus reniformis Linford and Oliveira, on cotton in Louisiana. Such effects

on reniform nematode reproduction may be direct due to the production of fungal metabolites

or indirect via fungal mediated alterations in cotton seedling physiology. Laboratory

investigations described herein attempted to test these hypotheses.

Materials and Methods

A single egg mass of R. reniformis isolated from cotton ( Gossypium hirsutum L.) roots

collected from Morehouse Parish, Louisiana was propagated on Rutgers tomato in a

greenhouse with temperatures ranging from 22-35 C. Nematode and egg inoculum was

obtained from these stock cultures. Preadult females used as inoculum for the infectivity studies

were recovered from soil by a modified centrifugal-sugar flotation technique (3.8). The fungus

R. solani was isolated from cotton seedlings exhibiting seedling blight symptoms in a cotton field

in Richland Parish. Procedures for isolation, maintenance, as well as production of fungal

inocula on oat grains were as described in chapter 2. The experimental design used in all the

tests was a randomized complete block and data were analyzed using the SAS General Linear

Model procedure (3.20). Data for repeated experiments were pooled and analyzed.

42

Egg hatch studies

Experiment-! : The objective of this experiment was to study the effects of different

concentrations of culture filtrate (CF) of R. solani on hatching of eggs of R. reniformis. Cultures

of R. solani were grown on 30 ml of potato dextrose broth (PDB) in 250 ml Erlenmeyer flasks

inoculated with a single 1-cm-d potato dextrose agar (PDA) disc cut from the growing edge of a

3-day-old fungal culture. Flasks were incubated at room temperature (22-25 C) for 7 days and

the filtrate was separated from the mycelium by passing through Whatman No. 1 filter paper.

Filtrates were sterilized by vacuum filtration through a 0.45-pm-pore filter (Micro Filtration

Systems, Dublin, CA). Fungal extract thus obtained was designated as 100% and further

dilutions were prepared by adding sterile deionized distilled water (SDDW). Eggs of R.

reniformis were extracted by the NaOCI extraction technique (3.7). Eight-tenth ml of the 100%

filtrate was pipetted into polystyrene cell wells (Corning, NewYork) containing 0.2 ml of the

nematode egg suspension (40-50 eggs/well) to give a final concentration of 80% filtrate. Eggs

were incubated in 80,40, and 20% CF and in SDDW. There were a total of four treatments each

replicated five times. Eggs were incubated at room temperature (22-25 C) and hatched juveniles

were counted at 3-day intervals until hatching ceased. Data are expressed as percent

cumulative egg hatch (CEH). That is, total numbers of juveniles observed at each interval / total

numbers of eggs at day zero x 100. The experiment was repeated once. The pH of the different

concentrations of the CF, SDDW and PDB was determined.

Experiment 2 : This experiment was conducted to determine whether or not PDB used

for growth of R. solani had any influence on egg hatching. Procedures for collection of filtrates,

egg extraction, and incubation were as described for experiment 1. Eggs were incubated in

80% filtrates. Treatments consisted of nematode eggs incubated in filtrates obtained from

cultures of R. solani produced in PDB or SDDW. Filtrates from flasks containing similarly aged

PDB or SDDW minus fungus served as controls. Egg hatching in cell wells containing 40-50

eggs was monitored at 2-day intervals. Each treatment was replicated five times and the

experiment was repeated once.

Experiment 3 : The purpose of this experiment was again to monitor the effect of filtrate

of R. solani on egg hatching. In this experiment, however, the fungus was grown in

compartmentalized dishes (100 x 15 mm, Fisher scientific, Houston, TX) commonly referred to

as “ I plates One half (side 1) of each dish contained 24 ml of PDB and the other half (side 2)

contained 24 ml of SDDW. A single 1-cm-d PDA disc cut from the growing edge of a 3-day-old

culture of R. solani was introduced into side 1 and was allowed to grow over the SDDW of side

2. After 7 days, the mycelium was carefully removed from side 2 and discarded, the aqueous

contents were removed, filter sterilized as described previously, and added to cell wells

containing nematode eggs (40-50/well). Side 1 of control plates received PDA discs minus

fungus and contents were collected after 7 days from side 2. Contents were then filter sterilized

and added to cell wells containing eggs. Treatments were replicated five times, egg hatching

was monitored at 2-day intervals, and the experiment was repeated once.

Experiment 4; This experiment was conducted to determine whether or not exudates

from cotton seedlings infected with R. solani influence egg hatching. Cotton (Deltapine 90)

seedlings were produced in greenhouse germination trays containing a 3:2:1 mixture of methyl

bromide treated loamy soil (80.8% sand, 4.7% silt, 14.5% clay, pH adjusted to 6.5), autoclaved

sand, and Weblite (Weblite Corp., Roanoke, VA). After 15 days, seedlings were removed from

the trays , the root system was washed twice in SDDW, and seedlings of uniform weight (1.25 ±

0.10 g) were selected.

A sterile glass slide (7.5 cm x 2.5 cm) was placed inside a sterile Petri dish (15 x 1.5 cm)

and one end of the slide was elevated by placing a sterile glass rod (0.75 cm-d x 7.5 cm long)

beneath it. A single seedling was then placed onto the slide with the cotyledonary portion facing

the elevated end. The entire dish was then inclined 10 degrees by placing another glass rod

under the dish. Five ml of SDDW (pH 5.5) was then pipetted into the bottom of the dish where it

pooled and covered the seedling root system. A total of 20 Petri dishes were used for the

experiment. Seedlings in half of the dishes were inoculated by placing an oat grain colonized by

R. solani onto the glass slide next to the hypocotyl region of the seedling. Seedlings in control

plates received noncolonized oat grains. A single piece of sterile Whatman No. 1 filter paper (15

cm-d) was folded in half, moistened with 3 ml of SDDW and placed in the lower end of each lid to

provide moisture for fungus growth. Dishes were incubated at ambient temperature (22-25 C)

under supplemental light from plant Grow-Lux fluorescent bulbs (ca.16 pE. m-2. sec-1, at bench

surface) which provided a 14:10 hr light:dark photoperiod each day. After 24 hr, Petri dishes

were opened and the filter paper was remoistened with additional 2 ml of SDDW. After 48 hr,

symptoms of Rhizoctonia infection were apparent on inoculated hypocotyl tissue of seedlings

and filter papers were removed from all dishes. After 4 days, lengths of lesions on inoculated

plants were measured and the aqueous contents of all dishes were removed and pooled into

two samples representing Rhizoctonia infected or noninfected plants. The volume of each

sample was adjusted to 30 ml by the addition of SDDW and the pH was determined. Samples

were then filter sterilized by passing through a 0.2-pm-pore filter (Nalge Company, New York).

To collect nematode eggs, tomato roots infected with R. reniformis were washed, and

cut into 7-10 cm lengths. From the root segments, 50-60 individual egg masses were removed

with the aid of a stereomicroscope and placed into an autoclaved test tube. Five ml of 0.5%

NaOCI were then added to the tube and it was shaken vigorously for five minutes. Under a

laminar flow hood, the egg suspension was poured over a sterile 45-pm-pore (325 mesh, 7.5

cm-d x 3.75 cm-deep) sieve, nested in a 25-pm-pore (500 mesh, 7.5 cm-d x 3.75 cm-deep)

sieve fitted to the bottom of a storage dish (80 x 100 mm, Fisher Scientific, Lexington, MA).

Eggs were removed from the 500 mesh sieve by rinsing with SDDW. One-tenth ml of the egg

suspension (50-60 eggs) was pipetted into polystyrene cell wells containing 0.9 ml of one of the

following: filtrate from infected seedlings, filtrate from noninfected seedlings, a SDDW control

(pH 5.5), another control-SDDW adjusted to the pH of filtrates from infected and noninfected

seedlings (pH 6.7) using 5-mM phosphate buffer (pH 7.0). Each treatment was replicated six

times. Eggs were incubated at 25 C and hatched juveniles were counted at 2-day intervals until

hatching ceased. The experiment was repeated once.

45

Infectivity studies

Experiment 5 : The objective of this experiment was to study the infectivity of preadults

of R. reniformis on cotton seedlings parasitized by R. solani. Root systems of 15-day-old

seedlings of cotton (DP 90) produced under greenhouse conditions as described above were

washed in SDDW and seedlings of uniform weight (1.39 ± 0.24 g) were selected.

Twenty-five grams of methyl bromide-treated soil mixture was weighed and 15 grams

was layered at the bottom of a sterile Petri dish (15 x 1.5 cm). One cotton seedling was placed in

each dish so that the root system was spread across the layer of soil and the remaining 10 grams

of soil was used to cover the root system. Thirty-two such dishes were prepared and divided into

two groups. Seedlings in one group were inoculated with R. solani by placing an infested oat

grain next to the hypocotyl at the soil line. Each seedling in the second group received a

noninfested oat grain. Then each seedling was inoculated with 100 preadult females by

pipetting aqueous suspension containing the nematodes onto the soil surface. The soil was

wetted with 2.5 ml of SDDW, and dishes were covered and incubated as described for

experiment 4.

Soil was wetted with an additional 2.5 ml of SDDW after 48 hr. Four seedlings from each

group were harvested at intervals of 24, 48, 72, and 96 hr after inoculation. Infectivity was

monitored by counting females present on the entire root system with the aid of a

stereomicrocope. To monitor the presence of the fungus, hypocotyl lesion lengths were

measured at each harvest interval. This experiment was repeated once and an additional

treatment was included. The additional treatment consisted of wounding the hypocotyl of

another group of 16 seedlings by scraping a 5 cm region of the hypocotyl with a sterile razor

blade to induce wounding.

Results

E x p e r im e n t: There was an inverse linear (P < 0.0001) relationship between

concentrations of CF and CEH from day 3 to day 15 (Table 3.1). CEH at 15 days after initiation

was 36.5% in SDDW compared to 22.7%, 33.3%, and 35.7% in 80%, 40%, and 20% filtrates

46

Table 3.1. Response of eggs of Rotylenchulus reniformis to culture filtrates of Rhizoctonia solani.

Days after initiation

Treatment f 3 6 9 12 15

CF 80% 1.5* 5.9 14.2 19.6 22.7CF 40% 3.2 13.4 22.3 29.9 33.3CF 20% 3.8 15.5 24.8 31.7 35.7SDDW 7.1 17.5 26.4 33.2 36.5

ContrastLinear *** *** *** *** ***

Quadratic NS NS * *** **

t CF = culture filtrate. SDDW = sterile deionized distilled water. * Data are percent cumulative egg hatch at each interval and are composite means of two trials of the same experiment. *, **, *** = significant at P = 0.05, 0.01 and 0.0001 based on Ftest respectively. NS = nonsignificant.

47

respectively. Egg hatching ceased after 15 days. The pH values for 80%, 40%, 20% CF, and

SDDW were 7.2, 7.1, 7.1, and 5.4 respectively. The pH of PDB was 5.2.

Experiment 2 : Numbers of juveniles observed were influenced by culture medium (P <

0.0001) at each interval (Table 3.2). PDB inhibited egg hatching. On day 14, CEH in PDB was

52% less than that in SDDW. Filtrates obtained from media which contained the fungus

increased hatching of R. reniformis eggs from day 6 to day 14 (P < 0.0001). On day 14, CEH in

filtrates collected from media containing R. solani was 45.1% compared with 39.4% in filtrates

which contained no fungus and the increase was 14%. The interaction of culture medium and

fungus influenced CEH (P < 0.0001) from day 6 to day 14. Examination of individual treatment

means for day 14 (Fig. 3.1), revealed that CEH in SDDW with and without the fungus was 57.3%

and 56.9% respectively . However, CEH in filtrates collected from PDB inoculated with R. solani

was 50% greater than the corresponding PDB filtrate minus fungus (21.9% vs 32.9%). Similar

trends were apparent at earlier observation intervals.

Experiment 3 : The influence of fungal filtrate on CEH was not significant at any of the

observation intervals (Table 3.3). CEH on day 14 in filtrates obtained from the SDDW side of

compartmentalized “ I plates “ with the fungus was 48.5% compared with 50.2% in SDDW

filtrates collected from controls.

Experiment 4: At 4 days, the lesion length on the hypocotyls of seedlings inoculated

with R. solani was 38.9 ± 3.4 mm. There were no differences in the numbers of juveniles

observed in the root exudates of fungus infected and noninfected seedlings and water controls

2 days after the experiment was initiated (Table 3.4). On day 4, there were no differences in the

numbers of juveniles present in wells containing root exudates of Rhizoctonia infected and

noninfected seedlings. Also, CEH in water controls did not differ from that which occurred with

exudates from seedlings which received only a noncolonized oat grain. From day 6 to day 14,

significant increases were observed in egg hatching in root exudates compared with water

controls. The numbers of juveniles which hatched from eggs in exudates from seedlings with

Table 3.2. Response of eggs of Rotylenchulus reniformis to Rhizoctonia solani and growth medium.


Treatment Level 4 6 8 10 12 14

Medium (M) PDBf 0 .4 * 11.7 19.8 24.3 26.2 27.4SDDW 6.5 25.1 43.1 47.1 52.6 57.1

Fungus(F) -F 3.3 17.2 29.7 33.2 37.0 39.4

Source+F 3.6 19.5 33.3 38.1 41.8 45.1

Medium *** *** *** -*** *** ***

Fungus NS *** *** *** *** ***

M xF NS *** *** *** *** ***

t PDB = potato dextrose broth. SDDW = sterile deionized distilled water. -F = absence of fungus. +F = presence of fungus. $ Data are percent cumulative egg hatch at each interval and are composite means of two trials of the same experiment.*** = significant at P = 0.0001 based on Ftest. NS = nonsignificant.

Cum

ulat

ive

egg

hatch

(%

)

5 0 -

4 0 -

3 0 -

20 -

10 -

+FF*-F +F**PDB SDDW

Fig. 3.1. Means for the cumulative egg hatch of Rotylenchulus reniformis in experiment 2, for the interaction between medium x fungus. Vertical lines delimit standard errprs of means. *-F = absence of fungus. +F = presence of fungus. **PDB = potato dextrose broth. SDDW = sterile deionized distilled water.

CO

Table 3.3. Response of eggs of Rotylenchulus reniformis to filtrates of Rhizoctonia solani produced over water culture in compartmentalized “ I plates “.


Treatment Level 2 4 6 8 10 12 14

Fungus -F t 0 .2 t 7.9 21.8 32.1 40.0 46.3 50.2

Source+F 0.3 9.6 20.6 31.8 39.2 45.5 48.5

Fungus NS NS NS NS NS NS NS

t -F = absence of fungus. +F = presence of fungus, t Data are percent cumulative egg hatch at each interval and are composite means of two trials of the same experiment. NS = nonsignificant based on Ftest.

Table 3.4. Hatching of eggs of Rotylenchulus reniformis as influenced by root exudates of Deitapine 90 cotton seedlings inoculated and noninoculated with Rhizoctonia solani.

Treatment


2 4 6 8 10 12 14

No fungus (pH 6.7) 0.7a* 14.5ab 32.9a 48.7a 57.6a 60.9a 61,2aFungus (pH 6.7) 0.6a 16.1a 33.9a 50.2a 60.6a 63.7a 64.0aSDDW (pH 6.7) 0.5a 13.2b 23.9b 40.3b 47.9b 50.6b 50,9bSDDW (pH 5.5) 0.9a 12.1b 23.6b 40.6b 49.8b 52.5b 52.8b

t SDDW = sterile deionized distilled water. $ Data are percent cumulative egg hatch at each interval and are composite means of two trials of the same experiment. Means in columns followed by the same letter are not significantly different (P < 0.05) according to Duncans multiple range test.

52

and without R. solani did not differ. Juveniles recovered from the two water controls of pH 5.7

and 6.7 did not differ.

Experiment 5: Lesion lengths on hypocotyls of seedlings infected with R. solani were

0,11.9 ± 1.9 mm, 17.8 ± 2.1 mm, and 23.6 ± 2.3 mm respectively at the 24, 48, 72, and 96 hr

intervals. At 24 and 48 hr, there were no differences in the numbers of preadult females which

entered the root systems of fungus infected and noninfected seedlings (Table 3.5). At the 72

and 96 hr intervals, however, the numbers of infective preadults which entered root systems of

Rhizoctonia infected seedlings were significantly greater than the numbers which penetrated

root systems of seedlings which were not infected with R. solani. At 24 and 48 hr, numbers of

females observed on the root systems of wounded, Rhizoctonia infected and noninfected

seedlings did not differ (Table 3.6). At 72 and 96 hr, numbers of females recovered from the

root systems of seedlings infected with R. solani were significantly greater than the numbers

from both wounded and non Rhizoctonia infected seedlings. Numbers of females on root

systems of wounded and non Rhizoctonia infected seedlings did not differ.

Discussion

The greenhouse portion of this research documented increased reproduction by R.

reniformis on cotton infected with R. solani. Compared with non-fungus infected counterparts,

cotton plants parasitized by R. solani supported greater egg production and had greater

numbers of juveniles in soil. The laboratory component of this project attempted to clarify

whether or not R. solani influenced directly or indirectly the egg and/or infective life stages of R.

reniformis. Four experiments were conducted with eggs and each was repeated once. Three

experiments involved the fungus in the absence of a host and one was conducted in the

presence of the cotton host plant.

Filtrates from cultures of R. solani produced on potato dextrose broth inhibited the

hatching of eggs of reniform nematode. However, we can not discount a possible pH effect on

egg hatching in experiments 1 and 2. The pH of the 80% culture filtrate was 7.2 compared with

5.4, and 5.2 for SDDW, and PDB respectively. In experiment 1 the notion that the medium

53

Table 3.5. Influence of Rhizoctonia solani infection on infectivity of Rotylenchulus reniformis on Deltapine 90 cotton.

Hours after initiation

Treatment 24 48 72 96

No fungus 8 .1 a t 13.8a 20.0b 28.6bFungus 7.5a 14.3a 32.0a 47.0a

t Numbers of sessile females per root system. Values are composite means of two trials of the same experiment. Means in columns followed by the same letter are not significantly different (P < 0.05) according to Duncans multiple range test.

Table 3.6 Influence of Rhizoctonia solani infection and hypocotyl wounding on infectivity of Rotylenchulus reniformis on Deltapine 90 cotton.

Hours after initiation

Treatment 24 48 72 96

No fungus 7 .0a f 11.8a 18.3b 26.5bFungus 6.3a 13.3a 30.5a 44.8aWounded}: 5.6a 12.3a 20.0b 28.0b

t Numbers of sessile females per root system. Means in columns followed by the same letter are not significantly different (P < 0.05) according to Duncans multiple range test, t wounding accomplished by scraping a 5 cm region of the hypocotyl with a sterile razor blade.

54

inhibited egg hatch prompted the inclusion of a PDB control in experiment 2. The significant

interaction between fungus x medium confirmed the suggestion that R. solani indirectly

influenced egg hatching by reducing the toxicity of PDB rather than by directly by producing

compounds which increased egg hatching. A similar effect was observed by Metha et al. (3.14)

with R. solani and M.javanica. They found that potato dextrose broth was more inhibitory to the

hatching of eggs of M.javanica than the filtrates of R. solani. Rambir Singh et al. (3.18) also

observed that filtrates of R. solani grown on PDB were inhibitory to the hatching of the eggs of

root knot nematode, Meloidogyne javanica (Treub) Chitwood. However, both these reports

provided no information about the possible effect of pH of the CF on nematode egg hatching.

Attempts to produce cultures of R. solani over water for the purpose of eliminating

substrate effects on egg hatching were much improved by using “ I plates “. Inspite of the fact

that there was abundant mycelial growth, however, none of the experiments showed effects of

fungus on egg hatching. If the fungus does produce compound(s) which directly affect egg

hatching, they are not produced over water culture or concentrations produced are below

levels necessary to affect egg hatching.

Egg hatch data from experiment 4 showed clearly that root exudates from cotton

seedlings have a pronounced effect on nematode egg hatching. Although other investigators

(3.9) have shown that malvaceous hosts produce exudates which enhance hatching of reniform

nematode eggs, our work with cotton constitutes the first report of this type for cotton. The fact

that there were no differences in egg hatching between the two pH regimes (5.5 and 6.7)

tested suggests that a pH effect in our system was minimal. Literature relating to pH effects on

nematode egg hatching are variable. Work with root knot nematode (Meloidogyne incognita

(Kofoid & White) Chitwood) and the effect of pH on egg hatching indicated that pH influenced

egg hatching (3.11). Conversely, similar work with cyst nematode ( Heterodera glycines

Ichinohe) eggs indicated little, if any, pH effect on eggs (3.10).

Since we consistently found no fungus effect on egg hatching, experiments were

initiated to evaluate whether or not the infectivity of preadults, which could account for both

55

enhanced egg production and soil populations, were influenced by the fungus. In two repeated

experiments, increased numbers of preadult females were observed after 72 hr in the root

systems of Rhizoctonia infected seedlings. Wounding had no detectable influence on

infectivity of preadults. This observation constitutes the first report of Rhizoctonia enhanced

infectivity of reniform nematode for cotton; and, is one of the few reports of this phenomenon

in literature for any crop. Infectivity studies by Edmund (3.3) with P. penetrans and F. oxysporum

on alfalfa showed enhanced penetration by second stage juveniles within five days of fungal

infection. The time frame corresponds closely with our findings for R. reniformis and R. solani. In

subsequent work with the P. penetrans / F. oxysporum system, increased attractiveness of P.

penetrans to alfalfa roots was attributed to the release of more CO2 , resulting from fungal

infection (3.4). It is well known that pectinolytic and cellulolytic enzymes play a major role in the

pathogenicity of R. solani on cotton hypocotyls. (3.2, 3.22). Increased respiratory rate, which

would result in increased CO 2 evolution, in bean ( Phaseolus vulgaris L.) hypocotyl tissues

infected with R. solani has been reported by Bateman and Daly (3.1). It is likely, then, that

changes in the physiology of Rhizoctonia infection of the cotton hypocotyls affects root

metabolism, CO 2 production, and subsequent attractiveness of the roots to nematodes. Also,

Riddle and Bird, (3.19) in their chemotaxic assays, demonstrated that juveniles of R. reniformis

oriented their movement toward inorganic salts such as MgCl2 and NaCI. It is possible that, as the

result of enhanced root parasitism, quantitative changes in electrolyte concentrations in the root

exudates render root systems more attractive to nematodes. Van Gundy et al. (3.21)

documented this with M. incognita, which like R. reniformis is a sedentary endoparasite, as they

showed that the nematode induced leakage of exudates from tomato roots which contained

high concentration of Mg and Na.

At this point in time it has not been resolved whether the Rhizoctonia enhanced

infectivity of R. reniformis was direct or indirect since the fungus was in both the hypocotyl

tissue and soil. The increased egg production and enhanced soil population densities

56

consistently observed in our greenhouse studies in the presence of R. solani probably resulted

from enhanced infectivity of preadults.

Summary

The effect of culture filtrates of Rhizoctonia solani and the influence of root exudates of

R. solani infected cotton seedlings on hatching of eggs of R. reniformis was evaluated.

Infectivity of preadults of reniform nematode to cotton seedlings infected with R. solani was also

monitored. Filtrates of R. solani obtained from potato dextrose broth were inhibitory to the egg

hatching of R. reniformis, and the culture medium was more inhibitory to the egg hatching than

the filtrate. Filtrates of R. solani collected from sterile deionized distilled water did not affect the

egg hatching. Exudates from roots of cotton seedlings increased the hatching of eggs of R.

reniformis. Root exudates from R. solani infected and noninfected seedlings did not differ in

their effect on egg hatching. Infectivity of preadult females to cotton seedlings, however, was

significantly enhanced by the fungus and this probably accounts for most of the enhanced egg

production and soil population densities observed in greenhouse studies.

57

Literature Cited

3.1. Bateman, D. F. and J. M. Daly. 1967. The respiratory pattern of Rhizoctonia-infected bean hypocotyls in relation to lesion maturation. Phytopathology 57:127-131.

3.2. Brookhouser, L. W ., J. G. Hancook, and A. R. Weinhold. 1980. Characterization of endopolygalacturonase produced by Rhizoctonia solani in culture and during infection of cotton seedlings. Phytopathology 70:1039-1042.

3.3. Edmunds, J. E. 1964. Effect of Trichoderma viridae and Fusarium oxysporum upon ingress of alfalfa roots by Pratylenchus penetrans. Phytopathology 54:892 (Abstr.).

3.4. Edmunds, J. E., and W. F. Mai. 1967. Effect of Fusarium oxysporum on movement of Pratylenchus penetrans toward alfalfa roots. 57:468-471.

3.5. Faulkner, L. R., and W. J. Bolander, 1959. Interaction of Verticillium dahliae and Pratylenchus minyus in Verticillium wilt of peppermint: Effect of soil temperature. Phytopathology 59:868-870.

3.6. Faulkner, L. R., and C. B. Skotland. 1965. Interactions of Verticillium dahliae and Pratylenchus minyus in Verticillium wilt of peppermint. Phytopathology 55:383-386.

3.7. Hussy, R. S., and K. R. Barker. 1973. A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Disease Reporter 57:1025-1028.

3.8. Jenkins, W. R. 1964. A rapid centrifugal flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692.

3.9. Khan, F. A. 1985. Hatching response of Rotylenchulus reniformis to root leachates of certain hosts and nonhosts. Revue de Nematologie 8:391-393.

3.10. Lehman, P. S., K. R. Barker, and D. Huisingh. 1971. Effects of pH and inorganic ions on emergence of Heterodera glycines. Nematological 7:467-473.

3.11. Loewenberg, J. R., T. Sullivan, and M. L. Schuster. 1960. The effect of pH and minerals on the hatching and survival of Meloidogyne incognita larvae. Phytopathology 50:215- 217.

3.12. McGawley, E. C., M. C. Rush, and J. P. Hollis. 1984. The occurrence of Aphelenchoides besseyi in Louisiana rice seed and its interaction with Sclerotium oryzae in selected cultivars. Journal of Nematology 16:42-46.

3.13. McKeen, C. D., and W. B. Mountain. 1960. Synergism between Pratylenchus penetrans and Verticillium albo-atrum R & B in egg plant wilt. Canadian Journal of Botany 38:789- 794.

3.14. Metha, N., K. K. Wallia, and D. C. Gupta. 1990. Effect of culture filtrates of Rhizoctonia solani and Rhizoctonia bataticola cultured on different media on hatching of Meloidogyne javanica larvae. Plant Disease Research 5:96-99.

58

3.15. Mountain, W. P., and C. D. McKeen. 1962. Effect of Verticilium dahliae on the populations of Pratylenchus penetrans. Nematologica 7:261 -266.

3.16 Overstreet, C., and E. C. McGawley. 1988. Influence of Calonectria crotalariae on reproduction of Heterodera glycines on soybean. Journal of Nematology 20:457-467.

3.17. Overstreet, C., E. C. McGawley and J. S. Russin 1990. Interactions between Calonectria crotalariae and Heterodera glycines on soybeans. Journal of Nematology 22:496-505.

3.18. Rambir Singh, D. C. Gupta, and K. K. Wallia. 1986. Effect of Rhizoctonia solani culture filtrate on hatching of Meloidogyne javanica larvae. Indian Phytopathology 39:624-625.

3.19. Riddle, D. L., and A. F. Bird. 1985. Responses of the plant parasitic nematodes Rotylenchulus reniformis, Anguina agrostis and Meloidogyne javanica to chemical attractants. Parasitology 91:185-195.

3.20. SAS Institute Inc. 1989. SAS User’s Guide, Version 6, First Edition, Cary, NC: SAS Institute Inc., 479 pp.

3.21. Van Gundy, S. D., J. D. Kirkpatrick, and J. Golden. 1977. The nature and role of metabolic leakage from root-knot nematode galls and infection by Rhizoctonia solani. Journal of Nematology 9:113-121.

3.22. Weinhold, A. R., and J. Motta. 1973. Initial host responses in cotton to infection by Rhizoctonia solani. Phytopathology 63:157-162.

3.23. Winchell, K. L., and E. C. McGawley. 1991. Interactions of Hoplolaimus galeatus, Fusarium oxysporum, and Macrophomina phaseolina on Centennial soybeans. Nematologica 36: 401 (Abstr.).

CHAPTER 4

CONCLUSIONS

59

60

Results of this research indicate that the fungus Rhizoctonia solani Kuhn, incitant of

cotton seedling blight, influences reproduction of the reniform nematode Rotylenchulus

reniformis Linford and Oliveira. Colonization of cotton hypocotyl tissue by R. solani resulted in

increased egg production on roots. Augmented egg production ultimately resulted in increased

numbers of juveniles in soil. Enhanced reproduction of the nematodes was observed to be

consistent across isolates of R. solani, populations and inoculum levels of R. renifomis and

cotton cultivars. The nematode did not influence the severity of seedling blight and first

negatively impacted plant growth at 90 days. R. solani, however, caused significant reductions

in plant growth in greenhouse experiments. An inverse linear relationship was observed

between nematode inoculum levels and plant growth response. The interaction between the

reniform nematode and the seedling blight fungus on plant growth was antagonistic at 90 days.

Since R. solani parasitizes the hypocotyl region of cotton seedling and R. reniformis infects the

lateral roots, the influence of the fungus on nematode reproduction may be indirect via altered

plant metabolism or direct via interaction with the soil-associated portion of the fungal

population.

Investigations were carried out under laboratory conditions to evaluate whether or not

the metabolites of R. solani have a direct influence on the egg hatching of R. reniformis. Culture

filtrates (CF) of R. solani produced on potato dextrose broth were inhibitory to the egg hatching

of the nematode. However, the medium was more toxic to eggs than the filtrate. CF of R. solani

collected from sterile deionized distilled water did not affect egg hatching. Root exudates from

cotton seedlings stimulated egg hatching. Egg hatching was not influenced by the root

exudates from seedlings infected with R. solani. This suggests that the fungus has neither

direct nor indirect effects on egg hatching. Colonization of cotton hypocotyls by R. solani

enhanced the infectivity of preadult females of R. reniformis. The enhanced infectivity of

reniform nematode preadults to cotton seedlings infected by the fungus may be either direct or

indirect.

Areas for Future Research

One of the possible explanations for the enhanced reproduction of Rotylenchulus

reniformis observed in the presence of Rhizoctonia solani in cotton was via altered host

physiology. Research detailing the mechanisms of interaction between nematodes and fungi in

disease complexes is lacking and such investigations will lead to a better understanding of the

disease complexes. Results presented in this dissertation have generated the following

questions:

1. If the increased infectivity of reniform nematode preadults to Rhizoctonia infected seedlings

is indirect, is it due to increased release of gaseous or nongaseous exudates?

2. If the increased infectivity is due to gaseous exudates, is it possible to conduct such studies

in vitro by raising R. reniformis on callus tissue in axenic cultures, as was done with the

Pratylenchus penetrans / Fusarium oxysporum system?

VITA

Ambalavanan Sankaralingam was born on August 15,1957 at Rajavallipuram, TN, India.

He received his Bachelor of Science degree in Agriculture from Annamali University, TN, India

during 1978. He was ranked second among his fellow graduates. He started his graduate study

at Tamil Nadu Agricultural University (T. N. A. U.), TN, India and received his Masters degree in

Agriculture (Plant Pathology) in 1980. He received Sri. P. S. Jivanna Rao Medal for the

outstanding student in the Department of Plant Pathology during 1978-80. He was also

awarded the ASPEE Agricultural Research and Development Foundation Award to do his

doctoral studies; however, he decided to enter into a job. He joined as Assistant Professor

(Plant Pathology) at the same institute (T. N. A. U.) where he completed his masters program. He

was primarily involved in teaching and research. He was married to Muthulakshmi

Balasubramanian in August 1983. He is now the father of two girls viz., Gomathi and

Ulaganayaki. Sankaralingam was awarded the Louisiana Methodist World Hunger Scholarship

and he was deputed by his institute in August 1989 to do his Ph. D. at the Department of Plant

Pathology and Crop Physiology at Louisiana State University. During 1990, he was one among

the recipients of Watumull Estate Scholarship awarded to outstanding students of Indian origin

at L. S. U.

62

DOCTORAL EXAMINATION AND D ISSERTATIO N REPORT

C a n d id a te : A m balavanan S ankara lingam

M a jo r F ie ld : P la n t H ealth

T i t l e o f D is s e r t a t io n : The In te rre la tio n s h ip s o f R o ty len ch u lu s re n ifo rm is

L in fo rd an d O liv e ira w ith R h izocton ia solani K uhn on

Cotton

Approved:

f

J ■■ 7 'De^n o f th e G ra d u a te Schoo l

EXAMINING COMMITTEE:

m Jjth cl . Qu Uy_J!- ___

D a te o f E x a m in a tio n :

March 31, 1993

Documents

The Interrelationships of Rotylenchulus Reniformis Linford