Pathogenicity In order to confirm pathogenic nature of isolated fungal culture, seedling of cotton variety NHH- 44 was raised in pots in glass house/screen house. These seeds were sown in sterilized soil: compost: sand mixture (2:1:1) at the rate 5 seeds/pot (30 cm diameter) on germination 2 seedlings per pot were maintained. These seedlings were inoculated at the stage of 4-6 true leaves with spore suspension (2x10 6 spores/ml.). Inoculation was carried out by spraying the suspension with an automizer. For this purified culture was multiplied in conical flask [250ml containing sterilized PDA broth (100 ml/flask)]. These flasks were kept on mechanical shaker for 72 hrs at slow speed. This growth of fungus was then used for inoculation. Before inoculation, leaves were injured by rubbing carborandum powder to have small injuries for development of symptoms. Immediately pots were watered and entire seedlings with pots were covered with polyethene bags for 48 hrs to maintain humidity. Intermittently pot were watered and the polythene bags were also taken out for a few minutes to avoid rise in temperature. Observations were recorded by observing the plants daily and sufficient number of untreated control was maintained for comparisonSignificance:1. Potential impact ofAlternaria macrosporaon cotton production in West TexasDr. Jason E. Woodward, Mr. Aaron S. Alexander, Dr. Randal K. Boman, and Dr. Terry A. Wheeler. Texas A&M University, 1102 East FM 1294, Lubbock, TX 79403 Alternaria macrosporaZimm. is a widespread foliar pathogen of cotton (Gossypium hirsutumL.) found throughout most production regions of the world. The objectives of this study were to evaluate the impact ofA. macrosporaon various aspects of cotton production. Samples were collected from six locations to compare cotton yield and fiber quality between healthy and infected areas. Seed and lint yields, and selected fiber properties were significantly lower, and leaf grades significantly higher from infected areas. As a result the overall crop value was reduced by approximately $732 ha-1when infected withA. macrospora. Additional experiments are currently being conducted to examine the potential for transmission ofA. macrosporaon seed, and to evaluate the efficacy of selected seed treatments on growth and development ofA. macrospora. Results from this study will provide cotton producers with valuable information that can be used to manage this disease more efficiently in the future. Damage 1. Relative effects of Alternaria alternata and A. macrospora on cotton crops in Israel In large commercial fields, the ratio ofAlternaria macrosporato A.alternatalesions was 1:0-4 in cv. Pima and 1:19 in cv. Acala. The frequency ofA. alternataon Pima and ofA. macrosporaon Acala increased in an experimental field with mixed Pima and Acala plots. In both cultivars disease was inhibited by fungicidal treatment and by removal of flowers. For all parameters measured (leaf area, number of leaves, flowers and bolls, and yield), the responses to treatments were greater in Pima than in Acala.2. Influence of foliar application of nitrogen and potassium on alternaria diseases in potato, tomato and cottonABSTRACTThe hypothesis that enrichment of the foliage with nitrogen and potassium may enhance host resistance toAlternaria and thus reduce disease severity, was examined for potato and tomato (A.solani) and for cotton (A.macrospora). First, the activity of urea (CO(NH2)2) and the salts NH4NO3, (NH4)2S04, KNO3, KCl, K2SO4 and KH2P04 againstA. solani andA. macrospora was determinedin vitro; each of the compounds tested had a weak inhibitory effect on spore germination of A.solani (ED50 > 1000 g/l) and on mycelial growth of both A.macrospora andA. solani (ED50 > 10,000 g/l). Next, the effect of foliar application of selected nutrients onA. solani andA. macrospora was evaluatedin vivo on detached leaves of tomato and cotton. The diameter of A.solani lesions on leaves sampled from tomato plants treated with KNO3 was significantly smaller (by 56.5%) than that recorded on leaves sampled from untreated plants.A. macrospora severity on leaves sampled from cotton plants treated with urea was significantly lower than that observed on untreated leaves(70.8% reduction) but KNO3 did not affect disease severity significantly. The following step was to examine the effects of foliar application of ureaand KNO3 onAlternaria development in field experiments, two with potato and one with cotton. Foliar application of both nutrients (8-10 spraysin total) did not affectAlternaria severity as compared with the untreated control in any of the experiments. The fungicides chlorothalonil and tebuconazole,on the other hand, significantly suppressed the disease throughout most of the growing season. A mixture of urea or KNO3 with the fungicides did not further improve the effects of the latter when applied alone. Based on these results, it wasconcluded that foliar application of urea or KNO3 does not affect host response toAlternaria.

Way to control the fungus Tuesday, September 11, 2007 - 5:00 PMDevelopment of sensitive molecular diagnostic tools for detection of economically important fungal pathogens of cottonDr. P. K. Chakrabarty, Mr. R.L. Chavhan, Ms. S.V. Sable, Mr. A.V. Narwade, Dr. D. Monga, and Dr. B.M. Khadi. Central Institute for Cotton Research, Post Bag No 2., Shankar Nagar, P.O., Nagpur -440010, India1PCR protocols for detection and differentiation of strains ofRhizoctonia solani,R. bataticola,Ramularia areolaandAlternaria macrospora, four economically important fungal pathogens of cotton were developed. Based on nucleotide sequence of the internal transcribed spacer regions of ribosomal RNA genes of these pathogens four sets of primers were developed. Primers pRSol and pRBat were specific to strains ofR. solaniandR. bataticolaand supported amplifications of rDNA fragments of 255 and 400 bp, respectively. Primer pRare indiscriminately detected four strains ofR.areolaisolated from each of the only four cultivated species of cotton by supporting amplification of an universal amplicon of 372 bp. Strains ofA. macrosporacould be identified by amplification of a DNA fragment of 542 bp using primer pAmac and differentiated from other species ofAlternariaby PCR-RFLP of the rDNA product withBanII,HaeIII andMseI restriction endonucleases.Cultivated cotton (Gossypium arboreum,G. herbaceum,G. hirsutumandG.barbadense) in India suffers from large number of diseases that affect both above and underground parts of the plant causing considerable losses in quality and yield (Hillock, 1992; Srinivasan, 1994; Chakrabarty and Mayee, 2004). Besides bacterial and viral diseases, fungal diseases provide a real challenge to successful cultivation of cotton. Fungal foliar diseases such as Grey mildew, which is caused byRamularia areola, was predominantly a pathogen of diploid cotton (G. arboreumandG.herbaceum) which now infects tetraploid cotton (G. hirsutumandG. barbadense) as well (Mukewar et al. 1994). The disease causes extensive defoliation and has become a menace in central and South India. Leaf spot and blight caused byAlternaria macrosporais another destructive foliar disease of cotton that affects production of cotton in different regions (Srinivasan, 1994). Root rot and wilt, caused by the soil-borne fungal plant pathogens,Rhizoctoniaspp. andFusarium oxysporumf.sp.vasinfectum, are two major diseases that exist in different cotton growing regions of the country(Srinivasan 1994; Monga and Raj, 2003).Identification of the cause and prevalence of a disease is very essential for adequate and timely plant disease management, which in turns depends on accurate diagnosis and early detection of the pathogen. Often it may be desirable to examine the soil for prevalence of any potential pathogen even before the crop is sown. Early detection enables one to make decisions regarding cultivar choice and chemical control that can be used most effectively to prevent development of a potential plant disease epidemic (Ward et al. 2004). Diagnosis of the causal agent is also important for studies on epidemiology (NOTE: Some diseases and/or declines have been studied without knowing the biological cause such as Para wilt of cotton (Raj et al. 1991), yield loss relationships and designing new strategies for disease management. Traditional or classical methods of disease diagnosis and pathogen identification could be relatively slow, often requiring skilled taxonomists to reliably identify the pathogens at the genus or species level. Delays are damaging when quick diagnosis is needed so that appropriate disease control measures may be taken to prevent plant injury especially when high value cash crops like cotton and other important plant species are at stake.JOURNAL OF COTTON SCIENCE, Volume XXX, Issue XXX, 2007Advances in Biotechnology have intensified efforts in recent years to develop novel methods for detection and identification of plant pathogens. Nucleic acid has increasingly been used in recent years to develop diagnostic assay for plant pathogens (Ward et al. 2004). Molecular approaches mainly the polymerase chain reaction have been used widely as the tool for detection of fungal pathogens (Martin et al. 2000, Schaad and Frederick, 2002). Rapid PCR assay based on amplification of sequence of internal transcribed spacer (ITS) region of rDNA or pathogenicity genes have been developed and used for detection of several plant pathogens (Henson and French 1993). Molecular techniques, if not alone, can be used in conjunction with classical methods where the latter approaches can at least narrow pathogen diagnosis to genus level. Once genus is narrowed by morphology, symptomatology, host-specificity, etc., then PCR can be used to differentiate species.We developed PCR based diagnostic methods to detect strains ofR. solani,R. bataticola,Ramularia areolaandA. macrosporaMATERIALS AND METHODSFungal strains and maintenance. The sources of fungal species for which diagnostic tools were developed are given in Table 1. Fungal strains, except that ofR. areola, were grown and maintained on potato dextrose agar (PDA). For long term storage, they were stored in mineral oil at 4oC in 15 ml screw-capped Corning glass tubes. For DNA isolation the fungal strains except that ofR. areolawere grown in potato dextrose broth (PDB). PDB (100 ml) was inoculated with a 5 mm diameter plug of culture agar cut from the edge of 5 days old culture of each isolate grown on a Petri dish. The inoculated broth was incubated at 28 2oC for 7 days.Isolation of genomic DNA. The mycelial mat was filtered through Whatman No 1 filter paper and dried at room temperature. The genomic DNA was extracted from fresh mycelium by a modified DNA extraction protocol (Chakrabarty 2004). Approximately 0.5 g of dry mycelial mat was transferred to a clean sterile mortar. Added 1.5 g of White quartz sand (HiMedia, India), 2.5ml extraction buffer (100 mM Tris, pH 8.0, 20 mM EDTA, 0.5 M NaCl, 1% SDS, 0.5Mglucose) and 1.25 ml buffer saturated phenol/chloroform/isoamyl alcohol (25:24:1) at pH 8. The mixture was ground thoroughly with a pestle and the homogeneous slurry was transferred into several microfuge tubes using a wide-bore tip and centrifuged at 13,000 rpm for 5 min at room temperature. The aqueous phase from each tube was transformed to 1.5 ml microfuge tubes to a volume of 750l and re-extracted with equal volume of chloroform/isoamyl alcohol (24:1). The contents of the tube were mixed by inverting several times followed by centrifugation at 13000 rpm for 5 min. The aqueous phase was again transferred to a new tube and the DNA was precipitated with 0.1 volume 3M sodium acetate (pH 5.2) and 1 volume isopropanol at room temperature for 10 min. The DNA was pelleted by centrifugation at 13,000 rpm for 10 min at 40oC, rinsed with 70% ethanol, and resuspended in 200 l of TE (10 mM Tris, 1 mM EDTA, pH 8.0) buffer containing 20 g/ml RNAse. Using this method, genomic DNA was extracted from strains ofA. macrospora,R. solani, andR. bataticola.Spores from the surface of the lesions of mildew infected leaves were scraped with a sterile tooth-pick moistened with sterile distilled water. The spore mass were boiled for 5 min and used as the template in PCR reaction.ITS-PCR and cloning of rDNA sequences. PCR amplification of rDNA sequences for all fungal species was conducted in 50 l reaction volumes using conserved ITS1 and ITS4 primers (White et al. 1990). Each reaction consisted of 2 l of 50 ng/l DNA template, 5 l of 10X PCR buffer, 0.5 l of 25mM dNTPs, 1.5 l of 15 mM MgCl2, 0.3 l of 1.25U Taq DNA polymerase, 1 l each of 10 M primers ITS1(5' TCC GTA GGT GAA CCT GCG G 3 ') and ITS 4 (5 TCC TCC GCT TAT TGA TAT GC 3 ') and 38.7l sterile distilled water. The PCR protocol was standardised to amplify rDNA sequences from a strain each ofR. solani, R. bataticola, A. macrosporaand four strains ofR. areolainfecting four cultivated species of cotton:,G. arboreum,G. herbaceum,G. hirsutumandG. barbadense. The standardised protocol had cycling parameters of initial denaturation at 94oC for 4 min followed by 33 cycles of denaturation at 94oC for 1 min, annealing at 55oC for 1 min and extension at 72oC for 1.5 min. A final extension at 72oC for 5 min was done at the end of amplification. Negative controls were used to test for false priming and amplification.A 10-l PCR amplification product for each of the fungal species was visualized in a 1%agarose gel and viewed under UV light following staining with ethidium bromide.Cloning of rDNA fragments. Gel purified fragments of ~ 650 bp comprising partial sequences of 18S and 28S rRNA genes, and complete sequences of ITS1, 5.8S and ITS2 of each fungal strain were cloned in pGEMT (Promega, Madison, WI, USA), following manufacturers protocol, unless stated otherwise. The ligation reaction was incubated overnight at 40oC. The ligation mix was transformed in Escherichia coli (XL-1 Blue) by heat shock method. The tube containing the competent cells (200 l) was removed from 70oC and allowed to thaw on ice. Ligation reaction mixture (2 l) was added to the tube of competent cells following incubation on ice for 5 min. The cells were subjected to heat shock at 42oC for 30 second and transferred on ice for 2 min. Heat-shocked cells were dispensed in 250 l LB in micro centrifuge tube. The transformation mix was incubated at 37oC in an orbital shaker at 220 rpm for 45 min to allow expression of the plasmid. The entire transformation mixture was then plated on LB agar containing Ampicillin (70ug/ml), Xgal (80 g/ml) and IPTG (50 M). The plate was incubated overnight at 37oC. The recombinant clones were identified by blue white colony selection. The white putative recombinant colonies were streaked on LB agar supplemented with Ampicillin (70 g/ml). The plasmid isolation from the putative transformants was done by the rapid miniprep protocol (Chakrabarty, unpublished). The recombinant clones were confirmed by digesting plasmid DNA withAatII andPstI.Sequencing of ITS amplicons and multiple alignment of sequence data. The cloned ribosomal RNA genes and the ITS regions of each fungal strains were sequenced using T7 and SP6 vector based primers at M/S Bangalore Genie Pvt. Ltd. Bangalore (India). The rRNA sequences of each fungal pathogens, comprising of partial sequences of 18S rRNA and 28S rRNA; and complete sequences of ITS 1, 5.8S rRNA and ITS 2 were submitted in GenBank. The DNA sequences of each accession were aligned among themselves as well as with other published sequences available in GenBank using BlastN and http://www.justbio.com.Development of species-specific primer and PCR detection protocol.Following multiple alignments of the rDNA sequences, regions of dissimilarity in ITS 1 and ITS 2 sequences were determined and used to design primers specific to three fungal species:R. solani, R. bataticolaandR. areolaand anAlternariagenus-specific primer forA. macrospora. To test specificity of primers in detecting strains of respective cotton pathogen only, the genomic DNA of each pathogen was subjected to PCR amplification with each set of primer. ForA. macrospora, sequence variability with respect to otherAlternariaspecies infecting other economically important plants was not good enough for designing species-specific primers. Therefore, restriction fragment length polymorphism analyses of amplified rDNA fragments with different restriction enzymes were used to differentiateA. macrosporafrom otherAlternariaspecies. PCR amplified ITS regions ofA. macrosporaand seven otherAlternariaspecies were digested with restriction enzymes viz.,BalI,BalII,BanII,ClaI,HaeIII,HindIII,HphI,MboI,MseI,NlaIV,SacI,TfiI,SalI,Sau3aI,SmaI,XhoI andXmaI. Restriction digestion reaction was carried out in 15 l volumes and consisted of 0.5 l restriction endonuclease (5U/l), 1.5l restriction buffer (10X), 11l sterile distilled water and 3l of PCR product. The digestion was carried out at 37oC for 2 h. The digested PCR product was resolved on 2 percent agarose gel, stained with ethidium bromide (0.5 g/ml) and visualized under UV to analyze nucleotide polymorphism in amplified fragment.RESULTSDNA based PCR diagnostic protocols were developed to identify four fungal pathogens of cotton, includingR. solani, R. bataticola, A. macrosporaandR. areola(Table 1). PCR Amplification of cotton fungal species with conserved primers ITS1 and ITS4 yielded an ~600 base pair rDNA product which were cloned in plasmid pGEMT (Fig. 1 a & b). Analysis of rDNA fragments from fungal strains revealed presence of partial sequences of 18S and 28S rRNA genes and complete sequences of ITS 1 and ITS 2 along with 5.8S rRNA gene. The sequences of the entire ITS 1/5.8S/ITS 2 regions together with short termini from large and small subunit genes, were obtained for each of the four pathogens. The sequences were deposited in GenBank and accession numbers obtained for each of them (Table 1). There was significant variation in the sequences of the ITS regions, especially within ITS1 and ITS 2, although several highly conserved regions were present in both regions. Regions of significant sequence variability inR. solani, R. bataticola, R. areolaandA. macrospora, were good enough to design species-specific oligonucleotide primers for strains of first three species. Four different sets of primers capable of differentially detecting these four pathogens were designed. The pathogens, the primers and the sizes of the diagnostic amplicons are given in Table 2.Primers pRsol, pRbat, pAmac and pRare could specifically detect strains ofR. solani, R. bataticola, A. macrosporaandR. areolaby amplification of rDNA fragments of 255, 400, 542 and 372 bp, respectively (Fig. 2). Primers pRsol and pRbat can specifically amplify strains ofR. solaniandR. bataticola, respectively. pRsol successfully detected strains ofR. solanitested but did not detectR. bataticolastrains infecting cotton (Fig. 3a). On the other hand pRbat could amplify a DNA fragment of 400 bp from strains ofR. bataticolabut not from the strains ofR. solanicollected from different cotton growing zones of the country (Fig. 3b).Primer pRare indiscriminately detected four strains ofR. areola, each isolated fromG. hirsutum, G. barbadense, G. arboreumandG. herbaceumby universal amplification of a DNA fragment of 372 bp. pAmac amplified a rDNA fragment of 542 bp from strains ofA. macrospora.Each set of primer supported amplification of the strains of respective target pathogen but failed to detect members of other three pathogens tested (Fig. 4a-d).The primer pAmac however, was not specific toA. macrosporaof cotton but supported amplification of the rDNA fragment from several species ofAlternaria, such asA. alternatastrains from sorghum and sunflower,A. longipes, A. porri, A. dianthicola, A. citriandA. brassicae(Kadam 2005). Lack of adequate variability in nucleotide sequences in the ITS region of different species ofAlternariadid not allow designing species-specific primers forA. macrospora. Strains ofA. macrosporacould however, be identified and differentiated by possession of two unique restriction endonuclease sites such asBanII andMseI in the rDNA repeat unit. These two enzymes sites are not present in any otherAlternariaspecies studied. There was a singleBanII site in the ITS1 region ofA. macrosporawhich cleaved the linear PCR amplified rDNA repeat into two fragments of 448 and 127bp size (Fig. 5). The rDNA region also possessed twoMseI sites one each in ITS2 region and 28S rRNA gene that generated three fragments of 418, 136 and 21bp size. Also unlike in allAlternariaspp. which possessed singleHaeIII site,A. macrosporahad twoHaeIII sites one each in ITS1 and ITS2 regions and generated three DNA fragments of 368, 140 and 67 bp. Besides, comparison of the rDNA sequences amplified using conserved ITS1 and ITS4 primers inA. macrosporaagainst other Alternaria species, revealed that the former has the highest number of nucleotides (575 bp) in the ITS region.DISCUSSIONFour sets of pathogen-specific primers developed as a part of this study enabled successful diagnosis of cotton-specific strains ofR. solani, R. bataticolaandR. areola, while PCR-RFLP method could differentiate strains ofA. macrosporafrom several other species of this pathogen.Detection of polymorphism using PCR-RFLP analysis of the ribosomal DNA- ITS region has been successfully used for identification of several species of fungi (Martin et al. 2000). This simple technique requires only minute amounts of DNA and two specific conserved primers flanking the ITS region of rDNA genes. This is one of the groups of genes most frequently targeted for phylogenetic studies and codes for rRNA. The main reasons for the popularity of rDNA are that it is a multicopy, non-protein-coding gene, whose repeated copies in tandems are homogenized by concerted evolution and is therefore treated as a single locus gene. Furthermore, the ribosomes are present in all organisms and ribosomal RNA genes are the most commonly used target for fungal and bacterial diagnostics (Ward et. al 2004). The amplified products of ITS region of 11 fungal species from different crops (Kadam 2005), including strains ofR. solani, R. bataticola, A. macrosporaandR. areolareported in the present study, ranged between 569-575 bp, coinciding with the sizes obtained from similar fungal pathogens from other strains of the same species. The multiple alignments of the rDNA sequences using sequences available in GenBank and sequences from this study revealed significant variability in ITS1 and ITS2 regions directly allowing us to design species-specific primers. Considerably greater sequence variations is found in the internal transcribed spacer (ITS) regions between the rRNA genes within a rRNA repeat unit (Henson and French 1993). Nazar et. al. (1991) found adequate sequence differences in the ITS regions of the cotton wilt fungi,Verticillium dahliaeandV. alboatrum, to design primers that specifically amplify the DNA of each species. Primers based on differences in ITS 1 sequences ofLeptosphaeria maculansallowed specific amplification of weak or virulent isolates of this fungal pathogen (Xue et. al. 1992). Specific primers were also designed and developed based on the ribosomal genes to detect and differentiate several species of the genusPhytophthora,an economically important fungal pathogen of crop plantsRistaino et. al. 1998; Appiah et. al. 2004).For fungal species such asAlternaria alternata, A. longipes, A. dianthicola, A. citri, A. brassicae, A. macrospora and A. porri, where significant variability in the nucleotide sequence of rDNA did not exist, inter and intra-specific variation was evaluated by analysis of the ITS region of rDNA using restriction fragment length polymorphism. Cleavage of amplified fragments with specific restriction enzymes revealed extensive polymorphism that allowed further differentiation of theseAlternariaspecies.A. macrosporapossessed certain unique restriction sites likeBanII andMSeI. The presence of these unique restriction sites are consistent with observed nucleotide sequence variability in the rDNA sequences of theseAlternariaspecies that included addition or deletion of several conserved nucleotides. Such substitutions are responsible for obliteration of some conserved restriction sites or creation of some unique sites. The inter-specific variation among several species ofPhytophthorainfecting cocoa could be clearly distinguished by restriction analysis of the PCR amplified rDNA regions with unique restriction enzymes (Appiah et al 2004). The PCR-RFLP analysis of rDNA-ITS region has also been successfully used for identification and differentiation of several species of ectomycorrhizal fungi (Amicucci et al.1996, Eliane et al. 2002). Previously, Chakrabarty et al (2005) developed PCR based diagnostic protocols for detection ofXanthomonas axonopodispv.malvacearumand cotton leaf curl virus, two major pathogens of cotton, by developing primers based on their pathogenicity genes. A PCR protocol for detection ofAlternaria radicinaon carrot seed was developed by Pryor and Gilbertson, (2001). The primers were designed based upon the sequence of a cloned RAPD fragment of the pathogen.The results obtained during the present investigation showed that the internal transcribed spacer regions of the ribosomal RNA gene sequences can be used to design species-specific diagnostic tools. Furthermore, the ITS-restriction fragment length polymorphism analysis has potential to serve as markers for differentiation of closely related species or the strains belonging to same species.REFERENCESAmicucci, A.; I. Rossi, L. Potenza, A. Zambonelli, D. Agostini, F. Palma, and V. Stocchi.1996. Identification of ectomycorrhizae from tuber species by RFLP analysis of the ITS region. Biotechnol. lett. 18: 821-826.Table 1: The fungal strains, sources and GenBank accessions of their ITS sequence.Sr. NoPathogen species/ strainSexual/ asexual formSourceGenBank Accession

1Rhizoctonia solaniKuhn.Thanatephorus cucumeris(Frank) DonkInfected rootsof cottonDQ339103

2Rhizoctonia bataticola(Taub.), ButlerMacrophomina phaseolina (Tassi) Gold.Infected rootsof cottonDQ339102

3Alternaria macrosporaZimm.-Infected leavesof cottonDQ156342

4Ramularia areolaAtk. (hirsutum)Mycosphaerella areola (Ehrlich and Wolf.)Mildewed leavesofG. hirsutumDQ459076

5R. areolaAtk. (barbadense)Mycosphaerella areola (Ehrlich and Wolf.)Mildewed leaves ofG. barbadenseDQ631897

6R. areolaAtk. (arboreum)Mycosphaerella areola (Ehrlich and Wolf.)Mildewed leavesofG. arboreumDQ459081

7R. areolaAtk. (herbaceum)Mycosphaerella areola (Ehrlich and Wolf.)Mildewed leaves ofG. herbaceumDQ459082

Table 2. The fungal pathogens, diagnostic primers and sizes of the amplified products.Sr. NoPathogen speciesPrimersamplicon (bp)

1Rhizoctonia solanipRsol255

2Rhizctonia bataticolapRbat400

3Ramularia areolapRare372

4Alternaria macrosporapAmac542

Fungicide treatment and varietal effects onAlternarialeaf spot of Pima cottonMary W. Olsen, Plant Pathology DepartmentLee Clark, Safford Agricultural CenterHal Moser, Maricopa Agricultural CenterAbstractThe effect of foliar treatments for prevention ofAlternarialeaf spot was evaluated in the field on six varieties of Pima cotton. Disease was significantly reduced by protective sprays of mancozeb and micronized sulfur but not by foliar applications of urea in trials at the University of Arizona Safford Agricultural Center in Safford, AZ.. Treatments had no significant effects on yields. Significantly fewer lesions developed on Pima variety UA 4 than on the other varieties. Disease pressure was relatively light, and even though scheduled preventive sprays with mancozeb were effective, fungicide applications probably would not increase yields under the environmental conditions of this experiment.IntroductionAlternarialeaf spot of cotton, caused by the fungusAlternariamacrospora, causes lesions on leaves, bracts, and bolls of cotton. Disease is common in Arizona only under very humid conditions, and is usually associated with the onset of rains in the summer months at higher elevations. In the Safford Valley and other cotton growing areas of Graham, Greenlee, Cochise, and Pima Counties, disease can be severe on Pima cotton, causing defoliation if rain and high humidity are persistent in July, August and September. Variations in susceptibility among cotton cultivars have been reported (Cotty, 1987). Pima variety S5 was more susceptible than S6, and both Pima varieties were more susceptible than DP90 or other upland varieties. There is no data available comparing susceptibility of newer Pima varieties. Although older leaves are believed to be more susceptible to infection, disease development is not related to plant age (Shtienberg, 1993). Late season infections usually are not considered a problem since yields are probably not affected.There are currently no fungicides registered for use on cotton for control ofAlternarialeaf spot in Arizona. In other cotton growing regions of the world where disease is a problem and causes yield losses, fungicides such as maneb, mancozeb, difenoconazole and tebuconazole are used as protectant sprays (Shtienberg, 1991, 1992). Fungicides may be applied as often as every ten days to two weeks and initiated before flowering. Because of the restricted occurrence ofAlternarialeaf spot in the United States, especially in the Southwest, it is unlikely that new fungicide labels will be forthcoming for disease control. Therefore, it is important to determine the efficacy of foliar treatments that have current labels on cotton for the control ofAlternarialeaf spot.The objectives of this study were to (1) determine if preventive sprays of candidate foliar treatments would reduce disease incidence significantly; (2) demonstrate varietal susceptibility of Pima cotton toAlternarialeaf spot; (3) determine the effect of disease on yield; and (4) generate data for effective foliar treatments that would lead to a label for use on cotton for control ofAlternarialeaf spot of cotton.Materials and MethodsThis study was conducted at the University of Arizona Safford Agricultural Center. Six varieties of Pima cotton were planted according to standard practices in 4 row plots 15 m long with 3 replications in a randomized block design. Varieties were OA 361, UA 4, OA 312, OA 325, S6 and S7. Varieties S6 and S7 are currently available for commercial use and are planted in the Safford Valley; varieties OA 361, OA 312 and OA 325 are short season varieties developed by Olvey and Associates and are well suited to the Safford Valley; UA 4 is a short season variety developed by the University of Arizona Pima Breeding group.Foliar treatments for disease control - mancozeb, sulfur, and urea - were selected on the basis of their potential availability for use and their cost. Mancozeb already has a registration on cotton for prevention of cotton rust and is registered for use on other crops for control ofAlternariadiseases; micronized sulfur was applied because of its combined fungicidal and insecticidal potential and current registration on cotton for mite control; and urea, a known greening agent, was evaluated since disease has been shown to be related to leaf age (Shtienberg, 1993). Mancozeb was applied as 1.75 lb/ac Penncozeb 75df, sulfur as 7.5 lb/ac Microthiol Special, and urea as 5 lb/ac foliar urea by ground spray application on August 15, September 1, and September 15, 1997. These three application dates at two week intervals were chosen since more than two or three applications are considered economically unfeasible at current cotton prices and the six week interval was considered reasonable for disease control based on other studies (Shtienberg, 1992 ). Control plots were not treated. Treatments were applied along the varietal plots in a split-plot design.Disease was assessed on September 19, 1997 by counting the number of lesions on leaves at the fifth node down from the terminal node. Ten leaves were sampled from each plot and taken to the laboratory whereAlternarialesions were counted. Lint yields were determined at harvest by mechanical harvesting of plots and assuming 35% lint. Data were analyzed using the General Linear Models Procedure and Duncan's Multiple Range Test of SAS.Results and DiscussionAs shown inTable 1, the number of lesions per leaf was reduced significantly by the application of mancozeb and sulfur compared to the urea treatment or untreated control. Mancozeb was the most effective treatment and reduced lesions by more than 50%. However, the average number of lesions per leaf, even in the untreated controls, was low. Environmental conditions were not favorable for disease in 1997, and disease pressure was relatively light compared to years with higher humidity and more rain in July and August. Treatments had no significant effect on yields (Table 1). There was a varietal effect on the number of lesions per leaf (Table 2). Variety OA 325 had significantly higher numbers of lesions per leaf than S-6 or UA 4, with UA 4 having significantly fewer lesions per leaf than all other varieties. OA 312 had a significantly higher yield than UA 4, but was not different from the other varieties.Results indicate thatAlternarialeaf spot will not reduce yields of Pima cotton under the environmental conditions of these trials, and foliar applications would not be warranted. However, treatments of Pima cotton should be repeated in years of higher disease pressure and at different application dates in order to compare higher lesion numbers on yields. Trials carried out when weather conditions are more conducive to disease development would give growers the information needed to decide if and when to make foliar treatments.Literature cited1. Cotty, P. J. 1987. Evaluation of cotton cultivar susceptibility toAlternarialeaf spot. Plant Disese 71:61082-1084.2. Shtienberg, D. and J. Dreishpoun. 1991. Suppression ofAlternarialeaf spot in Pima cotton by systemic fungicides. Crop Protection 10: 381-385.3. Shtienberg, D. 1992. Development and evaluation of guidelines for the initiation of chemical control ofAlternarialeaf spot in Pima cotton in Israel. Plant Disease 76: 1164-1168.4. Shtienberg, D., Y. Kremer and A. Dinoor. 1993. Influence of physiological age of Pima cotton on the need for fungicide treatment to suppressAlternarialeaf spot. Phytopathology 83: 1235-1239.

3. This is a part ofpublication AZ1006: "Cotton: A College of Agriculture Report," 1998, College of Agriculture, The University of Arizona, Tucson, Arizona, 85721. Any products, services, or organizations that are mentioned, shown, or indirectly implied in this publication do not imply endorsement by The University of Arizona. The University is an Equal Opportunity/Affirmative Action Employer.This document located at http://ag.arizona.edu/pubs/crops/az1006/az100610a.htmlReturn to Cotton 98 indexWhy homeopathic medicines are use for Alterneria

Alternaria leaf spot of cottonSubmitted by naipagropediaraichur on Thu, 17/05/2012 - 12:33 Posted inAlternaria leaf spot of CottonAlternaria leaf spot is one of major foliar disease.This disease occurs in almost all the cotton growing countries of the world. Hybrids are more susceptible to this disease.Disease infect onleaves resulting in suppression of plant growth and reduction of yield. High severity of the infection causes strong defoliation of cotton, sharp decrease of yield and crude fiber quality.EtiologyCausal organisum :Alternaria macrosporaConidia are light-brown, 22-27 x 9-11 . They affect cotton cotyledons in seedlings, also bolls and their fiber. Mycelium ofA. macrosporais dark-brown. Conidiophores are light brown, single or in groups. Conidia are red-brown, 90-180 x 15-22 in size.Disease cycleThe undecomposed crop residues and infected seeds provide the primary source of inoculum, giving rise to infected cotyledons, which support the early stages of an epidemic. Primary infection of lower canopy leaves can be initiated from conidia splashed up from infected crop residues or blown into the crop from other foci of infection.Alternaria spp., also attacks the bolls and grow on exposed lint if bolls open in wet weather, giving rise to contaminated seed. The disease cycle is completed when infected leaves fall to the ground.SymptomsSmall, pale to brown, round or irregular spotsLeaves become dry and fall off.Cause cankers on the stem.Infection spreads to the bolls and finally falls off. Brownish spots on leaves Symptoms on boll Severely infected field with Alternaria leaf spot EpidemiologyFavourable condition for pathogy was high humidity, intermittent rains and moderate temperature of 25-28OC. The pathogen survives in the dead leaves as dormant mycelium. The pathogen primarily spreads through irrigation water. The secondary spread is mainly by air-borne conidia.DiseasesAlternaria gossypii (Jacz.) Nisik., K. Kimura and Miyaw.; Alternaria macrosporaZimm. - Alternaria Leaf Spot of Cotton.Object mapSystematic position.Kingdom Fungi, phylum Ascomycota, class Ascomycetes, order Pleosporales, family Pleosporaceae, genus Alternaria.Synonyms.Macrosporium gossypii Jacz.; Alternaria longipedicellata Snowden.Biological group.Saprotroph.Morphology and biology.The causative agents of Alternaria Leaf Spot of Cotton (Macrosporium Leaf Spot) are fungi Alternaria gossypii and A. macrospora developing in their life cycle in only anamorphous stages. Mycelium of A. gossypii is dark brown. Conidiophores are brown, single or in groups. Conidia are light-brown, 22-27 x 9-11 mkm in. They affect cotton cotyledons in seedlings, also bolls and their fiber. Mycelium of A. macrospora is dark-brown. Conidiophores are light brown, single or in groups. Conidia are red-brown, 90-180 x 15-22 mkm in size. They affect leaves, bracts in seedlings and adult plants, bolls. The causative agents cause necroses on cotyledons, leaves, and bolls of cotton in form of dark-green and then brown, rounded or various shaped spots with clearly expressed zonality. At high humidity light pink or dark conidial sporulation appears on necrotic spots. Affected boll fiber is brownish-red. Systematic position of the causative agents of cotton alternariosis and macrosporiosis is sometimes considered indistinct; they may represent one or two different species (Gorlenko, 1968). Sources of the infection are affected cotton crop residues, seeds, and also weeds. Additional vectors of the infection are aphids parasitizing cotton plants.Distribution.The Alternaria Leaf Spot is spread on cotton everywhere in the Central Asian and Caucasian countries of the former USSR.Ecology.The causative agents affect cotton more intensively at high air humidity and at daily average temperature about 25C. Strong severity of the disease is observed on old leaves with slow metabolic process.Economic significance.At the moderate affection of cotton by the causative agents, assimilative processes are broken in leaves resulting in suppression of plant growth and reduction of yield. High severity of the infection causes strong defoliation of cotton, sharp decrease of yield and crude fiber quality. Gossypium barbadense cotton varieties are more strongly affected than varieties of Gossypium hirsutum. Control measures are crop rotations including alternation with cereals and unaffected crops, application of the biological preparation of Trichoderma, duly weed control in crops, the use of chemical control against the disease or aphids, if necessary.Reference citations:Gorlenko M.V. 1968. Agricultural phytopathology. Moscow: Visshaya Shkola, 434 p. (In Russian).CABI Bioscience Databases. 2004.http://www.SpeciesFungorum.org.Peresypkin V.F. 1974. Agricultural phytopathology. Moscow: Kolos, 560 p. (In Russian).Peresypkin V.F. 1987. Atlas of diseases of field cultures. Kiev: Urozhai, 144 p. (In Russian).Pidoplichko N.M. 1977. Fungi are parasites of cultural plants. Keys. Kiev: Naukova Dumka, Vol. 2. 299 p. (In Russian). Yakutkin V.I.Picture is taken from Peresypkin V.F. 1987. Atlas of diseases of field cultures. Kiev: Urozhai. Table 88.