Mycosphaerella areola—The Teleomorph of Ramularia areola of Cotton in Brazil, and Its Epidemiological Significance

While Ramularia leaf blight of cotton caused by Ramularia areola is of top most importance for Brazil, information is lacking regarding the survival mechanism of this pathogen during the cotton-free period. The teleomorph of R. areola is expected to belong to the genus Mycosphaerella. In the present study attempts were made to verify occurrence of this teleomorph in the State of Mato Grosso, Brazil. Decaying cotton leaves were collected two months after harvest of 2014 from 44 commercial and experimental fields where aerial fungicidal applications were made or not during the crop cycle to control the Ramularia leaf blight. Examination of the decaying cotton leaves revealed presence of abundant sclerotia, spermagonia and ascoma of Mycosphaerella sp. intermingled with each other during the cotton-free period in most of the leaf samples. Mono-ascospore isolations were obtained from the ascoma and considering their cultural, morphological, pathological and DNA sequence analysis they were identified as Mycosphaerella areola. M. areola and R. areola isolates produced similar symptoms under glasshouse inoculations. Reisolation of the pathogen from the symptoms produced by M. areola isolates yielded R. areola. Some cotton leaves showing such symptoms were kept on the soil surface on plastic trays for two months under natural field condition. After this period the decaying leaves showed abundant perithecia identical to their original M. areola. ITS rDNA sequence analyses revealed identical sequences from M. areola and R. areola isolates. Occurrence and the viability of the perfect stage M. areola during the cotton-free period on the left-over stubble from one season to another were interpreted as the survival mechanism of the pathogen and were considered responsible for the Ramularia blight epidemics in the State of Mato Grosso. Disease management practices for the State of Mato Grosso are discussed. This is the first report about the occurrence of the M. areola in Brazil.

Systemic Acquired Resistance of Cotton, Soybean and Common Bean to <i>Rhizoctonia solani</i>and <i>Sclerotium rolfsii</i>Induced by Shale Water Seed Treatment

Root rots of cotton, soybean and common bean caused by Rhizoctonia solani and Sclerotium rolfsii are basically soil-borne diseases and are difficult to control through the use of fungicides. One of the alternatives to control these diseases could be through the induction of Systemic Acquired Resistance (SAR). It is believed that shale water as a by-product obtained during the process of extraction of petroleum from fossil rock may act as an inducer of SAR to some pathogens of some crop plants. The objective of the present investigation was to verify the effect of seed treatment with shale water in inducing SAR to R. solani and S. rofsii root rots of cotton, soybean and common bean. Seed treatment experiments were conducted in the greenhouse on seedlings of these three crops using naturally or artificially infested soil with R. solani or with S. rolfsii. Treatments with seeds treated with shale water significantly reduced the average number of plants infected with the two pathogens. Consistent results were obtained in repeated experiments. SAR in cotton and common bean to R. solani varied between 86.16% and 91.13%, while for S. rolfsii in soybean and common bean varied between 84.0% and 57.54% and was long lasting. This is the first report giving strong indication of SAR of the three crops to R. solani and to S. rolfsii. Patent regarding this investigation is obtained with Petrobras, Brazil, under the number IVP 12/039.

Search for a Microsatellite Marker Linked with Resistance Gene to Xanthomonas axonopodis pv.malvacearum in Brazilian Cotton

The cotton cultivar DELTAOPAL is resistant under field as well as under glasshouse conditions to the Brazilian isolates of Xanthomonas axonopodis pv. malvacearum (Xam). Segregating populations derived from the cross between this cultivar and one susceptible cv. BRS ITA 90, were utilized to identify molecular marker linked with the resistance gene to Xam by “Bulk Segregant Analysis (BSA)”. Two hundred and twenty microsatellite (Single Sequence Repeat—SSR) primers were tested. The amplification products were visualized in polyacrylamide gels stained with silver nitrate. Only one primer was informative and showed polymorphism between the DNA of the parents and their respective bulks of homozygous F2 populations contrasting for resistance and susceptibility, and hence was used to analyze DNA of 120 F2 populations. The microsatellite primer yielded one band of 80 bp linked with the resistance locus, which was absent in the susceptible parent as well as in the bulk of the homozygous susceptible plants of the cross. The segregation ratio as determined by phenotypic analysis was 3R:1S. It is believed that the microsatellite marker was linked with the resistance locus and hence may offer new perspectives for marker assisted selection against the angular leaf spot disease of cotton. It is however, felt necessary to repeat the microsatellite analysis and make sure that the primer is tightly linked with the resistance locus and at the same time verify the genetic distance between the marker and the resistance locus.

Systemic Acquired Resistance of Soybean to Soybean Rust Induced by Shale Water

Shale water as a by-product obtained by Petrobras, Brazil, during the process of extraction of petroleum from fossil rock may act as an inducer of Systemic Acquired Resistance (SAR) to some plant pathogens. The objective of the present investigation was to verify the effect of seed treatment and foliar application with shale water in inducing SAR of soybean to soybean rust under greenhouse and field conditions. In greenhouse experiments, seed treatment alone with shale water significantly reduced the severity of soybean rust and the control efficiency after 11 and 14 days after inoculation was between 54.1% and 57.8%. Whereas seed treatment and only one foliar application with shale water the control efficiency due to SAR 14 and 11 days after inoculation was between 99.7% and 100%, respectively. Such treatments gave similar results under field experiments where the control efficiency of soybean rust was between 79.0% and 99.35% in shale water treated plots as compared to the untreated plots, in 2015. Consequently, this resulted in yield increase between 14.8% and 28.8% depending upon the seed treatment and foliar applications with shale water alone or in mixture with a fungicide. Seed health testing revealed lower number of seeds infected with some pathogens in treatments where either shale water or fungicide was used. Seed treatment and one foliar application were sufficient to induce SAR against soybean rust. This is the first report to demonstrate SAR of soybean to soybean rust induced by shale water. Patent regarding this investigation is deposited with Petrobras, Brazil, under the number EVP 14/022.

Spore Types and Spore Production of Ramularia areola for Screening Cotton Germplasm for Resistance

Spore production ofRamularia areolahas always been a difficult task. Brazilian isolates ofR. areolaproduce spores of variable size and shape. The typical spores are 3 septate, rarely 4 and 5 septate, together with abundant single celled oblong to round bodies—a phenomenon not reported earlier forR. areola. Budding of spores is a continuous process as observed in our isolates. By repeated culturing the pathogen either ceases to produce typical spores or loses its pathogenic character. To solve this problem, a technique was developed to produce large quantity of typical spores under laboratory conditions. Sporulating cultures produced on Petri plates containing V8 juice-agar were kept on the laboratory bench till they became dry and then stored at 5°C for reisolation and/or for production of fresh inoculum. In such dried cultures spores remain viable for a period of over 12 months, and hence isolates ofR. areolaoriginatedfrom different geographic regions can be maintained in sporulating form. Results of the present investigation would aid cotton breeders and pathologists in screening germplasm resistant toRamulariaand in other genetical studies under glasshouse conditions.

Identification of Phenotypic and Genotypic Variability among the Isolates of Ramularia areola of Brazilian Cotton

Genotypic and phenotypic variation among 16 isolates of Ramularia areola of Gossypium hirsutum collected from five different geographical regions of Brazil was studied through virulence spectrum on three cultivars in the glasshouse and through ERIC- and REP-PCR and ITS1-5.8S-ITS2 rDNA analysis. Difference in virulence spectrum and molecular analysis of some isolates was observed. ERIC- and REP-PCR showed similar results and revealed a high level of diversity among the isolates. A unique profile for both ERIC and REP was obtained for most isolates. On the other hand, the ITS rDNA analysis did not show different PCR-RFLP patterns. While some isolates differed among each other considering genotypic and phenotypic reactions, no clear cut evidence was found about the existence of genetic lineages of R. areola in Brazil. Identification of genetic variability among the R. areola isolates originated from different geographic regions would permit screening of Brazilian germplasm and achieve sources with a wide spectrum of resistance. This is the first report of the genotypic and phenotypic variability among the R. areola isolates originated from five cotton growing regions of Brazil.

Corynespora Leaf Blight of Cotton in Brazil and Its Management

In recent years, Corynespora leaf blight is on the increase in cotton and has become an important disease in the State of Mato Grosso, Brazil. It attacks several plant species including soybean. It is not yet known whether the same strain of this pathogen attacks both the crops. The objectives of the present investigation were to verify the genotypic and phenotypic variation between the isolates attacking cotton and soybean and to verify the sources of resistance in cotton. Differential response of 23 cotton genotypes was studied under glasshouse conditions using mixture of two randomly selected isolates of C. cassiicola of cotton in equal proportion. Genotypic variation among three cotton and two soybean isolates was studied using ERIC/REP-PCR and rDNA molecular techniques. With one exception, all the cotton genotypes tested so far were susceptible to C. cassiicola isolates of both cotton and soybean. Similarly, results of both molecular techniques indicated that the C. cassiicola isolates attacking cotton and soybean belong to the same strain of the pathogen in Brazil. Since Corynespora blight is a newly immerging disease of cotton and soybean in Brazil, integration of crop rotation and the sanitary practices are suggested to manage the disease.