UNIVERSITI PUTRA MALAYSIA

PRODUCTION AND EFFICACY OF Exserohilum Longirostratum AS A BIOHERBICIDE FOR THE CONTROL OF ITCH GRASS (Rottboellia

Cochinchinensis) IN SUGARCANE FARMS

CHARLES BORROMEO ALESTER A. ABI FP 2008 31

PRODUCTION AND EFFICACY OF Exserohilum Longirostratum

AS A BIOHERBICIDE FOR THE CONTROL OF ITCH GRASS

(Rottboellia Cochinchinensis) IN SUGARCANE FARMS

By

CHARLES BORROMEO ALESTER A. ABI

MASTER OF AGRICULTURAL SCIENCE

UNIVERSITI PUTRA MALAYSIA

2008

PRODUCTION AND EFFICACY OF Exserohilum Longirostratum

AS A BIOHERBICIDE FOR THE CONTROL OF ITCH GRASS

(Rottboellia Cochinchinensis) IN SUGARCANE FARMS

By

CHARLES BORROMEO ALESTER A. ABI

Thesis Submitted to the School of Graduates Studies, Universiti Putra

Malaysia in Fulfilment of the requirements for the

Degree of Master of Agriculture Science

September 2008

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To

Sinoe & Sama

Sikiea’ & Sinokoėn

Kumaa Iggy do Kuopusanku

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfillment of the requirement for degree of Master of Agricultural Sciences.

PRODUCTION AND EFFICACY OF Exserohilum Longirostratum AS A BIOHERBICIDE FOR THE CONTROL OF ITCH GRASS (Rottboellia

Cochinchinensis) IN SUGARCANE FARMS

By

CHARLES BORROMEO ALESTER ANAK ABI

September 2008

Chairperson : Associate Professor Dr. Jugah Kadir, PhD

Faculty : Agriculture

Itch grass or Rottboellia cochinchinensis (Lour.) W.D. Clayton is

considered as on of the most important weeds in tropical countries. Integrated

weed management is a tool-box of options that can be tailored to individual

farm, cropping and weed situations. Biological control is one of these

management options, therefore the production method and field efficacy of

Exserohilum longirostratum as a bioherbicide to control itch grass in

sugarcane was investigated. The growth and sporulation of E. longirostratum

was excellent in media strength at recommended rate (200ml V8juice + 800ml

H2O). The optimum pH for conidia production and conidia germination was in

the range of pH 6 – 7. The conidia stored in liquid suspension (water and oil)

and freeze-dry failed to germinate. Air dried conidia in powder formed stored

at 70C and 200C has germination rate of 80% and remained viable for 6

months in comparison to those stored at other temperature levels. The conidia

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stored at 1 % Relative Humidity (RH) (silica gel) remained viable for more

than 6 months with germination rate of 80%. Deterioration of conidia stored at

1% RH was slow (rL = -0.41064) compared to those stored at 96.5% RH using

K2SO4 saturated solution where the rate of deterioration was significantly fast

(rL = -0.73). Susceptibility of several sugarcane varieties towards E.

longirostratum were tested using 1x107/ml conidia concentration, and the

results indicated that all sugarcane varieties tested were resistant to E.

longirostratum as indicated by lower values of area under disease progress

curve (AUDPC) and decreasing rates of disease progress. Sugarcane variety

95R-1004 was selected for the field trials as this variety was newly introduced

to be planted at Federal Land Development Authorities (FELDA) in Chuping,

Perlis.

In this study variety 95R-1004 and itch grass at 6-8 leaf stage were

sprayed with 107,108 and 109 conidia/ml concentrations in 10% of oil

emulsion. Plants were sprayed three times at interval of 1 week. The three

applications treatment of E. longirostratum at 109 conidia/ml provided

excellent control (100% mortality) of R. cochinchinensis compared to the other

treatments as shown by higher AUDPC value (1168 units) and faster disease

progress rate (rL 0.61 unit/day). There was no significant difference in tiller

numbers and the growth of sugarcane between the inoculated plots and the

untreated control or plots treated with the fungus-free oil emulsion. The dry

weight of itch grass was highest when treated with fungus-free oil emulsion

(0.275kg) or untreated controls (0.290kg). None of the itch grass plants

survived in plots treated with chemical herbicide (BASTA®), or plots with E.

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longirostratum. The sugarcane dry weight was significantly high (1.6kg) in

plots treated with E. longirostratum at 109 conidia/ml compared to the other

treatments. This may be due to eradication of itch grass in this treatment

earlier than in the other treatments, thus there was no competition between

sugarcane and itch grass, resulting in a faster growth of the sugarcane plants.

This research indicated that E. longirostratum has an ability to provide

excellent control of R. cochinchinensis at 6-8 leaf stage under field conditions.

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Abstrak tesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains Pertanian

PRODUKSI DAN KECEKAPAN Exserohilum Longirostratum SEBAGAI BIOHERBISID UNTUK RUMPUT MIANG (Rottboellia Cochinchinensis) DI

KAWASAN PENANAMAN TEBU

Oleh

CHARLES BORROMEO ALESTER ANAK ABI

September 2008

Pengerusi : Profesor Madya Dr. Jugah Kadir, PhD

Fakulti : Pertanian

Rottbolliea cochinchinensis atau rumput miang adalah antara rumpai yang

sangat penting di Negara-Negara tropika. Pengurusan Rumpai Bersepadu

ialah satu pilihan kaedah yang boleh dipadankan terhadap perladang

persendirian, penanaman dan situasi rumpai. Kawalan biologi pula adalah

salah satu opsyen dalam pengurusan ini, Oleh sebab itu, produksi dan

kecekapan Exserohilum longirostratum sebagai pengawalan biologi rumpai ini

di kawasan penanaman tebu telah pun diselidik. Pertumbuhan dan sporulasi

E. longirostratum didapati sangat baik dalam konsentrasi media pada kadaran

yang disyorkan (200ml V8juice + 800ml H2O). Penghasilan dan percambahan

konidia kulat ini pula didapati sesuai dalam kadar pH6 hingga pH7.

Penyimpanan inokulum kulat ini dalam midium cecair (air dan minyak)

didapati tidak sesuai di mana tiada percambahan konidia direkodkan. Walau

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bagaimana pun, penyimpanan konidia kering udara dalam suhu 70C dan 200C

mampu memberi paras percambahan yang kekal sehingga 80% selepas

disimpan selama 6 bulan. Kelembapan relatif pada 1% yang dikekalkan oleh

jeli silika (Silica Gel) merupakan keadaan yang terbaik untuk penyimpanan

konidia kering dalam suhu bilik di mana tahap pertumbuhan mencapai 80%

selepas 6 bulan. Kerosakan konidia dalam keadaan penyimpanan 1%

kelembapan relative (rL = -0.41064) juga sangat perlahan berbanding dengan

penyimpanan dalam kelembapan tinggi (K2SO4 ; rL = -0.73). Kepekaan dan

kecederaan beberapa variati tebu terhadap kulat ini pada kosentrasi 1

x107/mL juga telah diperhatikan dan didapati, kesemua variati tebu resistan

terhadap E. Longirostratum seperti yang ditunjukkan pada nilai Kawasan

dibawah Keluk Perkembangan Penyakit (AUDPC) yang rendah. Walau

bagaimana pun, varaiti 95R-1004 telah dipilih untuk kajian selanjutnya di

lapangan kerana ia merupakan variati yang baru diperkenalkan untuk di

tanam di FELDA Chuping.

Kajian keberkesanan dilapangan telah di jalankan keatas rumput

miang dan tanaman tebu pada peringkat 6-8 daun dengan konsentrasi

konidia E. longirostratum pada 107, 108, 109 dalam formulasi 10% minyak

sawit dan 5% pelarut yang disembur pada selang seminggu setiap satu.

Penyemburan tanaman dilakukan sebanyak 3 kali kekerapan dengan

seminggu tempoh perantaraan memberi kesan pengawalan rumput miang

yang sangat baik (100%) berbanding rawatan lain, dan ditujukan pada nilai

AUDPC yang tinggi (11682) dan nilai perkembangan penyakit yang tinggi(rL =

0.619 unit/hari). Tidak terdapat perbezaan yang ketara pada pertumbuhan

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anak bilah dan perkembangan pertumbuhan tebu di antara plot yang dirawat

dan plot yang bebas dari rawatan E. longirostratum. Berat kering rumput

miang di dalam plot yang bebas rawatan didapati tinggi dan mempunyai

perbezaan yang ketara berbanding di kawasan plot yang dirawat. Tiada berat

kering rumput miang dapat direkodkan di kawasan rawatan E. longirostratum

dan BASTA® kerana rumput miang di kawasan ini terhapus sama sekali.

Walau bagaimanapun, berat kering tebu di kawasan rawatan E.

longirostratum pada 109 konidia/mL didapati tinggi dan mempunyai perbezaan

yang ketara berbanding kawasan rawatan lain dan kawasan bebas rawatan.

Besar kemungkinan keadaan ini berlaku disebabkan rumpai miang terhapus

lebih awal oleh rawatan E. longirostratum berbanding kawasan plot lain, maka

tiada persaingan antara tebu dengan rumpai tersebut menyebabkan tebu

tumbuh dengan cepat. Keputusan kajian ini menbuktikan E. longirostratum

mempunyai keupayaan pengawalan yang sangat baik terhadap rumput miang

pada tahap 6-8 daun di lapangan.

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ACKNOWLEDGEMENT

All thanks and praises to almighty God, Who are Truth and the Origin of all

knowledge for His blessing that enlighten my minds and strengthen my body,

and blessed be Jesus Christ our Lord for His thought and guidance that keep

me walk on the path of light.

A deep appreciation and gratitude to my supervisory committee especially

Assoc. Prof. Dr. Jugah Kadir as a chairman for his endless guidance, bright

ideas, helpful comments, his kind concerns and understanding. I would also

like to convey my extended gratitude to Assoc. Prof. Dr. Abdul Shukor Bin

Juraimi and Dr. Soetikno S. Sastroutomo for their invaluable advice and

supervision until the completion of this thesis.

Special thanks to the ever helpful En. Yusof, En. Din, En. Joe, En. Nazri and

Pn. Asmalina for their kind assistance. I would like to thank my fellow friends;

Mohd Helmi @ Ujey, Kevin Cheong, Adam Supu, Zaiton, Fitri, Saw Chin and

En. Ariffin for their help and moral support.

Infinite thanks to my beloved parents, brothers, sisters, nieces & nephews for

their love, prayers and understanding throughout this journey. Last but not

least, thank you to Iggy for her patience, caring support and concern.

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This thesis submitted to the senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Agriculture Sciences. The member the Supervisory Committee were as follows: JUGAH KADIR, Ph.D. Faculty of Agriculture Universiti Putra Malaysia (Chairman) Abdul Shukor Juraimi, Ph.D. Faculty of Agriculture Universiti Putra Malaysia (Member) Soetikno S. Sastroutomo, Ph.D. CAB International Southeast and East Asia Region (Member) -----------------------------------------------

HASANAH MOHD. GHAZALI, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date: 9 April 2009

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DECLARATION

I hereby declare that the thesis is base on my original work except for the quotations and citations which have been duly acknowledge. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions

CHARLES BORROMEO ALESTER ANAK ABI Date:

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TABLE OF CONTENTS Page DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGMENTS ix APPROVAL SHEETS x DECLARATION FORM xii TABLE OF CONTENTS xiii LIST OF TABLES xvi LIST OF FIGURES xviii LIST OF ABBREVIATIONS xx CHAPTERS I INTRODUCTION 1 II LITERATURE REVIEW 6 2.1 Itchgrass (Rottboellia cochinchinensis) 6 2.2 Bioecological Characterization 6 2.3 Distribution 10 2.4 Economic Importance 11 2.5 Management 13 2.5.1 Mechanical and cultural control 14 2.5.2 Chemical control 16 2.5.3 Biological control 18 2.5.4 Integrating tactics management 20 2.2 Biological control of weeds using Plant Pathogen 23 III MATERIAL AND METHODS 35 3.1 Introduction 35 3.1.1 Liquid phase culture procedure 36 3.1.2 Effect of pH on E. longirostratum mycelium production 36 3.1.3 Effect of pH on E. longirostratum conidia production 37 3.1.4 Effect of pH on E. longirostratum conidia germination 38 3.1.5 Effect of medium strength on conidiation of 38 E. longirostratum. 3.2 Formulation and storage 39 3.2.1 Viability of conidia in liquid formulation 39 3.2.2 Dried conidia Formulation 40 Freeze Drying Method 40 Air drying method 41 3.2.3 Effect of Relative Humidity during storage on viability of 41 dry conidia

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3.2.4 Effect of storage temperature on conidia viability 42 3.2.5 Data analysis 43 3.3 Field Delivery and Efficacy 43 3.3.1 Inoculums production 43 3.3.2 Susceptibility of sugarcane varieties to E. longirostratum 44 3.3.3 Field trials 44 3.3.4 Disease assessment and data analysis 46 IV RESULT AND DISCUSSION 47 4.0 Mass Conidia Production 47 4.1 Effect of pH on mycelial growth 47 4.2 Effect of pH on conidia production 48 4.3 Effect of pH on conidia germination 49 4.4 Effect of V8-juice concentration in the growth medium on 50 conidiation of E. longirostratum 4.5 Effect of storage method on conidia viability 52 4.5.1 Storage in liquid solution 52 4.5.2 Storage of dried conidia 53 4.5.3 Freeze drying method 54 4.5.4 Air dried conidia 54 4.5.5 Effect of relative humidity on viability of air-dried conidia 55 4.5.6 Effect of storage temperature on viability of air-dried conidia 57 4.6 Glass House mini trial 59 4.6.1 Effect of E. longirostratum on several Sugarcane Varieties 59 4.7 Field delivery and efficacy 62 4.7.1 Disease development on itch grass 62 4.7.2 Treatments effect on sugarcane 65

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V GENERAL DISCUSSION 70 VI CONCLUSION 75

REFERENCES 77

APPENDICES 93 Appendix A 93

Appendix B 96 Appendix C 97 Appendix D 102 Appendix E 104

BIODATA OF THE STUDENT 117

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LIST OF TABLES

Table Page Conidia storage in liquid formulation using paraffin oil and 53 water. Solutions were mix with different level of Dextrose as growth enhancer and result were obtained 0-2 weeks after storage in 00C. Effect of various RH on E. longirostratum conidia 56

viability Effect of different temperatures on viability of air-dried conidia 58 Effect of E. longirostratum on several Sugarcane Varieties. 61 ANOVA table of disease development on itch grass by 63 107,108 and 109 of conidia concentration as treatments in 3 frequencies of application Field Efficacy of E. longirostratum for controlling itch grass. 64 Itch grass post application comparison for all treatments by 64 number of panicles, dry weight (g) and tillers. ANOVA table of disease development on sugarcane by 107,108 67 and 109 of conidia concentration as treatments in 3 frequencies of application. Effect of E. longirostratum to sugarcane plants in field efficacy 68 trial for controlling itch grass Sugarcane post application comparison for all treatments by 68 number of panicles, dry weight (g) and tillers. Anova table on the effect of pH level on E. longirostratum 93 yield (Biomass) by dry weight Anova table on the effect of pH level on E. longirostratum 94 conidia production Anova table on the effect of pH level on E. longirostratum 95 conidia germination Anova table on the effect of media strength on E. longirostratum 96 conidia production

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Result on effect of freeze drying method on conidia viability 97 Anova table on the effect of desiccants intended for maintaining 98 RH condition on E. longirostratum viability. Anova table for the effect of storage temperature on 99

E. longirostratum conidia viability Anova table for disease development on several sugarcane 102 varieties cause by E. Longirostratum Anova table of disease development on itch grass 104 Anova table for number of itch grass panicles produced 1 107 month after E. longirostratum application. Anova table for total dry weight of itch grass 1 month 108 after E. longirostratum application Anova table for total number of itch grass tillers produced 1 109 month after E. longirostratum application Anova table of disease development on sugarcane 111 Anova table for sugarcane height 1 month after 112 E. longirostratum application. Anova table for total dry weight of sugarcane1 month after 114

E. longirostratum application Anova table for number of sugarcane tillers produced 1 month 115 after E. longirostratum application

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LIST OF FIGURE

Figure Page Rottboellia cochinchinensis morphology 9 The effect of pH on mycelium production of E. longirostratum 48 7 days after incubation Effect of pH on of E. longirostratum yield. 49 Effect of pH on conidia germination on Water Agar (WA) 24hrs 50 after room incubation Conidia production on different concentration of V8 Agar 51

Effect of medium (V8 Juice) strength on E. longirostratum 52 yield

The effect of RH on viability of air-dried conidia in the storage 56 environment

The effect of storage temperature on conidia germination of 58

E. longirostratum Reaction of several Sugarcane Varieties when spray 60 inoculated with E. longirostratum. Disease progress of Exserohilum longirostratum on 61 several sugarcane varieties evaluated by disease severity percentage. Disease progress of Exserohilum longirostratum on itch 63 grass seedlings in field efficacy trial. Effect of E. longirostratum on R. Cochinchinensis under 66 field condition. Disease progress of Exserohilum longirostratum on 67 sugarcane plants in field efficacy trial The effect of desiccants on conidia germination by purpose to 99 maintain Relative Humidity in the storage environment as interpret in Linear Logistic model.

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The effect of storage temperature on conidia germination 101 as interpret in Linear Logistic model. Disease progress of Exserohilum longirostratum on several 103 sugarcane varieties interpret by Linear Logistic model Disease progress of Exserohilum longirostratum on 106 itch grass seedlings in field efficacy trial as interpret in Logistic model Disease progress of Exserohilum longirostratum on 113 sugarcane seedlings in field efficacy trial as interpret in Logistic model

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LIST OF ABBREVIATION m2 Meter square

mm2 Millimeter square

% Percentage

PDA Potato dextrose agar

cm centimeter 0C Degree centigrade

mL milliliter

µl Microliter

SE Standard error rL Apparent rate values were obtain growth rate or epidemic rate

by transforming percentage data using logistic model.

R2 Square of multiple correlation

Vol Volume

DI Disease index

∑ Sum

HR Hypersensitive response

RH Relative humidity

pH Potential of Hydrogen

µ Micro

rpm Rotation per minute

SAS Statistical Analysis System

w/v Weight per volume

h Hour

AUGC Area Under Growth Curve

AUDPC Area Under Disease Progress Curve

Kg Kilogram

g gram

P Probability

NA Not Applicable

a.i Active Ingredients

ha Hectare

CHAPTER 1

INTRODUCTION

Rottboellia cochinchinensis (Lour.) W.D. Clayton (Poaceae) or itch

grass is an aggressive weed under various ecological conditions and has

been labeled as a major agriculture weed in many areas of the tropics and

subtropics infesting both annual and perennial crops. Its centre of origin was

believed to be from Africa and Asia, but was introduced into the New World at

the beginning of the century (Ellison and Evans, 1992). It is also a weed of

bananas, cassava, citrus, cowpeas, papayas, groundnut, pineapple, rice, and

sorghum in Cuba, Ghana, Jamaica, the Philippines, Trinidad and Venezuela

but there is little reference to it in cereal crops in North American cereal. It is

primarily a weed of warm-season crops in a variety of habitats around the

world but can also be found growing along roadsides and in other open, well-

drained sites and is an important species in old field succession (NAPPO

Bulletin, 2003).

Reproduction of R. cochinchinensis is by seed. In the Philippines, the

plant flowers all year long, and a single plant may produce 2000 -16000

seeds. Studies in Zimbabwe have shown that dense stands of the plant will

produce over 600 kg of seed per season (Holm et al., 1977). A single plant

can produce thousand of seeds over one growing season, and densities of up

to 500 plants /m2 have been recorded (Pamplona and Mercado 1991).

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Uncontrolled infestations of R. cochinchinensis were found to reduce

yield of white food corn by about 50% (Fisher et al., 1985). In addition to its

effect on crop yield, R. cochinchinensis is a problem to laborers, as the

needle-like hairs on the leaf sheaths break off in the skin and can cause

painful infections. It is also an alternative host of viruses causing corn leaf

gall and rice leaf gall (Agati and Calica 1949). In Malaysia R. cochinchinensis

causes yield losses of 60% in sugarcane yield. (Tan Teck Nee, 2004; per.

comm)

Land preparation and crop rotation can be the key for achieving good

cultural control. Manual control also has been practiced in several countries

but the cost of labour became a limitation (Chan et. al.,1990). Chemical

herbicides can give satisfactory kill of the weed, but cost (of both product and

application) and increasing incidence of herbicides resistance has become the

constrains. Most are not selective enough for use on the graminaceous crops,

which are mostly associated with this weed. The chemical does not persist

long enough in the soil to give control of the succeeding flushes of the

seedlings. Therefore, further studies on other alternate control are needed to

overcome this troublesome weed. One such alternative is the use of plant

pathogens which is often referred to as bioherbicides. Bioherbicides offer the

possibility of an inexpensive and environmentally benign means of weed

control through the utilisation of living organism to control or reduce the

population of an undesirable weed. The most important characteristics of a

bioherbicide are: easy to mass produced in vitro, high virulence, genetic

stability and restricted host range. In addition, fungi are capable of active

3

penetration of host tissue and infection is not dependent on vectors, natural

openings or wounds, which are required by bacterial and viral pathogens.

Thus, facultative fungal pathogens are the best candidates for spray

application.

Isolates of fungi collected from R. cochinchinensis have been screened

for host specificity and three of them were selected for further studies as

potential biological control agents. An isolate of Colletotrichum sp. from

Thailand was tested in the laboratory and in field trials as a possible candidate

for development as a mycoherbicide. The results were equivocal but a

synergistic response was found when a low dose of chemical herbicide was

added to the fungal inoculum (Ellison and Evans, 1993). Rust, Puccinia

rottboelliae, causes severe seedling infection in the field and preliminary host-

range tests with an isolate from Kenya suggest that it is specific to R.

cochinchinensis. Thus, this rust may have potential as a classical biological

control agent in the Americas, perhaps involving a management strategy

including early-season augmentation (Ellison, 1993). Investigations on

Sporisorium ophiuri, a systemic head smut, as a possible classical biological

control agent, suggested that it may have limited potential because of high

inoculum requirements; however, as the sporidia are readily produced in

culture, it may be possible to apply them to the soil as a form of

mycoherbicide (Reeder et al., 1999, Ellison and Evans, 1993). One of the

problems associated with S. ophiuri, is that it has only one disease cycle a

year and consequently, it has a slow intrinsic rate of spread within a

population of R. cochinchinensis. The other problem is it has very narrow

4

infection window that is it only infects R. cochinchinensis at flowering stage.

Seeds vigor may be reduced, however, this weed is also capable of

generating through rattons, and an infection of the seeds has little bearing on

the dispersal and survival of this weed.

A Curvularia sp. has been isolated from Trinidad and has been proven

to be highly damaging to R. cochinchinensis, while not damaging to rice,

sugarcane or pearl millet (Evans, 1999). It was able to kill R. cochinchinensis

in a few days; however it has a wide host range including maize (Ellison,

1992). Surprisingly few insects have been recorded attacking R.

cochinchinensis and only one unidentified gall midge was recorded in India

from R. compressa (Barnes, 1946). In East Africa a stem borer, a

lepidopteran leaf feeder and fly larva all proved to be non-specific

graminaceous feeder (Evans, 1999). In Malaysia, Azean (2004) has reported

the potential of Exserohilum longirostratum as bioherbicide for controlling this

weed and this fungus can be produced on V8-juice agar with a 12h light cycle.

However, the development of this pathogen as a bioherbicide requires that

various limitations in biological, technological, environmental, and economic

aspects to be solved. For example, effective methods to lower the cost of

mass production and formulation to improve the efficacy of the pathogen are

needed. Extensive studies are needed to develop methods of inoculum

production and to identify additives that prolong inoculum viability. Further

studies are required to confirm efficacy of this bioherbicide in commercial

fields.