Distribution and Sequestration of Cercosporin by Cercospora cf. flagellaris.

Plant-pathogenic fungi produce toxins as virulence factors in many plant diseases. In Cercospora leaf blight of soybean caused by Cercospora cf. flagellaris, symptoms are a consequence of the production of a perylenequinone toxin, cercosporin, which is light-activated to produce damaging reactive oxygen species. Cercosporin is universally toxic to cells, except to the cells of the producer. The current model of self-resistance to cercosporin is largely attributed to the maintenance of cercosporin in a chemically reduced state inside hyphae, unassociated with cellular organelles. However, in another perylenequinone-producing fungus, Phaeosphaeria sp., the toxin was specifically sequestered inside lipid droplets (LDs) to prevent reactive oxygen species production. This study hypothesized that LD-based sequestration of cercosporin occurred in C. cf. flagellaris and that lipid-inhibiting fungicides could inhibit toxin production. Confocal microscopy using light-cultured C. cf. flagellaris indicated that 3-day-old hyphae contained two forms of cercosporin distributed in two types of hyphae. Reduced cercosporin was uniformly distributed in the cytoplasm of thick, primary hyphae, and, contrary to previous studies, active cercosporin was observed specifically in the LDs of thin, secondary hyphae. The production of hyphae of two different thicknesses, a characteristic of hemibiotrophic plant pathogens, has not been documented in C. cf. flagellaris. No correlation was observed between cercosporin production and total lipid extracted, and two lipid-inhibiting fungicides had little effect on fungal growth in growth-inhibition assays. This study lays a foundation for exploring the importance of pathogen lifestyle, toxin production, and LD content in the pathogenicity and symptomology of Cercospora.

Alternative products to control late season diseases in soybeans / Produtos alternativos para o controle de doenças de final de ciclo na soja

In the last crop seasons, the complex of late season diseases (CLSD) of soybean (Glycine max L. (Merrill)), has been causing considerable reductions in the crop yield. Currently, there are no cultivars resistant to all pathogens that causes CLSD. The present study evaluated the effect of applying the acibenzolar-S-methyl resistance inducer, alternative products and fungicide on the severity of CLSD in the soybean cultivar BMX Potência RR during the 2013/2014 and 2014/2015 crops, in the field. The treatments for the experiments were: 1 - control (water); 2 - acibenzolar-S-methyl; 3 - calcium; 4 - micronutrients: copper, manganese and zinc; 5 - micronutrients: manganese, zinc and molybdenum; 6 - nitrogen-potassium fertilizer; 7 - Ascophyllum nodosum and 8 - azoxystrobin + cyproconazole with the addition of the adjuvant. Four applications of alternative products and two of fungicide were carried out in both harvests. A diagrammatic scale assessed the severity of CLSD at the phenological stage R7.1. The acibenzolar-S-methyl resistance inducer, alternative products (macro and micronutrients) and A. nodosum had no effect on the severity of CLSD in the two harvests. The fungicide (azoxystrobin + cyproconazole) reduced the severity of CLSD and prevented damage to productivity in both experiments.

Nas últimas safras, o complexo de doenças de final de ciclo (DFC) da soja (Glycine max L. (Merrill)), vem provocando reduções consideráveis no rendimento da cultura. Atualmente, não há variedades resistentes a todos os patógenos causadores das DFC. O objetivo do presente trabalho foi avaliar o efeito da aplicação do indutor de resistência acibenzolar-S-methyl, produtos alternativos e fungicida na severidade de DFC na cultivar de soja BMX Potência RR durante as safras 2013/2014 e 2014/2015, em campo. Os tratamentos para os experimentos foram: 1 - testemunha (água); 2 - acibenzolar-S-methyl; 3 - cálcio; 4 - micronutrientes: cobre, manganês e zinco; 5 - micronutrientes: manganês, zinco e molibdênio; 6 - adubo NK; 7 - Ascophyllum nodosum e 8 - azoxistrobina + ciproconazol com adição do adjuvante. Foram realizadas quatro aplicações dos produtos alternativos e duas do fungicida, nas duas safras. A severidade de DFC foi avaliada por escala diagramática no estádio fenológico R7.1. O indutor de resistência acibenzolar-S-methyl, os produtos alternativos (macro e micronutrientes) e A. nodosum não apresentaram efeito sobre a severidade das DFC nas duas safras. O fungicida (azoxistrobina + ciproconazol) reduziu a severidade das DFC e evitou danos a produtividade nos dois experimentos.

High-quality genome assembly of the soybean fungal pathogen Cercospora kikuchii.

Plant diseases caused by the Cercospora genus of ascomycete fungi are a major concern for commercial agricultural practices. Several Cercospora species can affect soybeans, such as Cercospora kikuchii which causes soybean leaf blight. Speciation in Cercospora on soybean has not been adequately studied. Some cryptic groups of Cercospora also cause diseases on soybean. Moreover, it has been known C. kikuchii population genetic structure is different between countries. Consequently, further genomic information could help to elucidate the covert differentiation of Cercospora diseases in soybean. Here, we report for the first time, a chromosome-level genome assembly for C. kikuchii. The genome assembly of 9 contigs was 34.44 Mb and the N50 was 4.19 Mb. Based on ab initio gene prediction, several candidates for pathogenicity-related genes, including 242 genes for putative effectors, 55 secondary metabolite gene clusters, and 399 carbohydrate-active enzyme genes were identified. The genome sequence and the features described in this study provide a solid foundation for comparative and evolutionary genomic analysis for Cercospora species that cause soybean diseases worldwide.

Effects of Purple Seed Stain on Seed Quality and Composition in Soybean.

Purple seed stain disease, caused by (Cercospora kukuchii), is a major concern in soybean (Glycine max (L.)) in Mississippi, USA, due to its effects on seed quality, reducing soybean seed grade and potential market price at elevators. Therefore, investigating the effects of purple seed stain (PSS) on seed quality (germination and vigor) and seed composition (nutrition) is critical. The objective of this study was to investigate the effects of PSS on seed harvest index, seed germination, seed vigor, and seed composition components (protein, oil, fatty acids, and sugars). A field experiment was initiated in 2019 in Stoneville, MS, at the Delta Research and Extension Center (DREC) on a Commerce silt loam soil (fine-silty, mixed, superactive, nonacid, thermic Fluventic Epiaquepts). Soybean variety Credenz 4748 LL was used. The results showed that infected (symptomatic) seed had a 5.5% greater Seed Index (based on 100 seed weight) when compared to non-infected (non-symptomatic, as control) seed. Non-infected seed had greater percent germination and seedling vigor when compared to infected seed. Germination was 30.9% greater and vigor was 58.3% greater in non-infected seed. Also, the results showed that infected seed with PSS had higher protein content and some amino acids. No changes in total oil and fatty acids. Sucrose and stachyose were lower in infected seed than in non-infected seed. The research showed that PSS impacted seed health and seed quality (germination and vigor) and seed composition (protein, sugars, and some amino acids). Purple stained seed should be avoided when planting and should be managed properly as low germination is a potential risk. Planting population should be adjusted accordingly due to lack of germination and vigor if PSS is present. This research help growers for purple seed management, and scientists to further understand the potential negative impact on seed quality and nutrition. Further research is needed before conclusive recommendations are made.

Gene expression during development and overexpression after Cercospora kikuchii and salicylic acid challenging indicate defensive roles of the soybean toxin.

KEY MESSAGE: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

Draft genome sequence data of Cercospora kikuchii, a causal agent of Cercospora leaf blight and purple seed stain of soybeans.

Cercospora kikuchii (Tak. Matsumoto & Tomoy.) M.W. Gardner 1927 is an ascomycete fungal pathogen that causes Cercospora leaf blight and purple seed stain on soybean. Here, we report the first draft genome sequence and assembly of this pathogen. The C. kikuchii strain ARG_18_001 was isolated from soybean purple seed collected from San Pedro, Buenos Aires, Argentina, during the 2018 harvest. The genome was sequenced using a 2 × 150 bp paired-end method by Illumina NovaSeq 6000. The C. kikuchii protein-coding genes were predicted using FunGAP (Fungal Genome Annotation Pipeline). The draft genome assembly was 33.1 Mb in size with a GC-content of 53%. The gene prediction resulted in 14,856 gene models/14,721 protein coding genes. Genomic data of C. kikuchii presented here will be a useful resource for future studies of this pathosystem. The data can be accessed at GenBank under the accession number VTAY00000000 https://www.ncbi.nlm.nih.gov/nuccore/VTAY00000000.

Elicitor-Based Biostimulant PSP1 Protects Soybean Against Late Season Diseases in Field Trials.

Currently, fungicide application in soybean production accounts for an important amount of global pesticide use, and it is therefore most desirable to find new healthier and more environmental friendly alternatives for the phytosanitary management in this crop. In this study, we present convincing evidence for effective induction of disease protection by the agricultural biostimulant PSP1, a formulation based on the plant-defense eliciting activity of the fungal protease AsES (Acremonium strictum elicitor subtilisin), in multiple field trials in Argentina. PSP1 was shown to combine well with commercial spray adjuvants, an insecticide, a herbicide and fungicides used in Argentinian soybean production without losing any defense-inducing activity, indicating an easy and efficient adaptability to conventional soybean production and disease management in the region. Results from multiple soybean field trials conducted with different elite genotypes at several locations during two consecutive growing seasons, showed that PSP1 is able to induce an enhanced pathogen defense which effectively reduced late season disease (LSD) development in field-grown soybean. This defense response seems to be broad-range as disease development was clearly reduced for at least three different fungi causing LSDs in soybean (Septoria glycines, Cercospora kikuchii and Cercospora sojina). It was noteworthy that application of PSP1 in soybean alone gave a similar protection against fungal diseases as compared to the commercial fungicides included in the field trials and that PSP1 applied together with a fungicide at reproductive stages enhanced disease protection and significantly increased grain yields. PSP1 is the first example of an elicitor-based strategy in order to efficiently control multiple fungal diseases under field conditions in the soybean crop. These results show the feasibility of using induced resistance products as complements or even full-good replacements to currently used chemical pesticides, fulfilling a role as important components of a more sustainable crop disease management system.

Validación de una técnica de Dot blot para la detección de Cercospora kikuchii en plantas de soja / Validation of the technique based on Dot blot for the detection of Cercospora kikuchii in soybean plants

Cercospora kikuchii, fitopatógeno usual en plantas de soja, ocasiona deterioro en la scosechas. Su identificación precoz y correcta evitaría el uso indebido de plaguicidas y permitiría iniciar un tratamiento adecuado. Una técnica rápida, económica y de fácil ejecución es el Dot blot, capaz de reconocer la presencia de una proteína específica del género conocida como CFP (Cercosporin Facilitator Protein). El objetivo de este trabajo fue validar dicha técnica para garantizar la fiabilidad del resultado. Para ello, se procesaron 29 plantas de soja infectadas y 31 plantas sanas teniendo en cuenta una confianza deseada del 95% y un error permitido del 5%. La técnica presentó una sensibilidad diagnóstica del 93,3% y una especificidad diagnóstica del 96,7%. La eficacia fue del 95% y los valores predictivos positivo y negativo del 96,6 y el 93,5%, respectivamente. Estos resultados la postulan como una herramienta útil para detectar precozmente c. kikuchii en plantas de soja.

Cercospora kikuchii is a common pathogen in soybean plants that causes crop spoilage. Its early and precise identification would prevent the misuse of pesticides and allow the initiation of an appropriate treatment. A quick, economical and easy-to-execute technique is the Dot blot, capable of recognizing the presence of a genus-specific protein called CFP (Cercosporin Facilitator Protein). The objective was to validate this technique to guarantee the reliability of the results. For that purpose, 29 infected soybean plants and 31 healthy plants were processed, taking into account a 95% desired confidence level and a permissible error of 5%. The technique provided a diagnostic sensitivity of 93.3% and a diagnostic specificity of 96.7%. The efficiency was 95% and positive and negative predictive values were 96.6% and 93.5%, respectively. These results postulate it as a useful resource for the early detection of C. kikuchii in soybean plants.

[Validation of the technique based on Dot blot for the detection of Cercospora kikuchii in soybean plants]. / Validación de una técnica de Dot blot para la detección de Cercospora kikuchii en plantas de soja.

Cercospora kikuchii is a common pathogen in soybean plants that causes crop spoilage. Its early and precise identification would prevent the misuse of pesticides and allow the initiation of an appropriate treatment. A quick, economical and easy-to-execute technique is the Dot blot, capable of recognizing the presence of a genus-specific protein called CFP (Cercosporin Facilitator Protein). The objective was to validate this technique to guarantee the reliability of the results. For that purpose, 29 infected soybean plants and 31 healthy plants were processed, taking into account a 95% desired confidence level and a permissible error of 5%. The technique provided a diagnostic sensitivity of 93.3% and a diagnostic specificity of 96.7%. The efficiency was 95% and positive and negative predictive values were 96.6% and 93.5%, respectively. These results postulate it as a useful resource for the early detection of C. kikuchii in soybean plants.

Changes occurring in compositions and antioxidant properties of healthy soybean seeds [Glycine max (L.) Merr.] and soybean seeds diseased by Phomopsis longicolla and Cercospora kikuchii fungal pathogens.

Changes in the compositions (isoflavone, protein, oil, and fatty acid) and antioxidant properties were evaluated in healthy soybeans and soybeans diseased by Phomopsis longicolla and Cercospora kikuchii. The total isoflavone content (1491.3 µg/g) of healthy seeds was observed to be considerably different than that of diseased seeds (P. longicolla: 292.6, C. kikuchii: 727.2 µg/g), with malonlygenistin exhibiting the greatest decrease (726.1 → 57.1, 351.9 µg/g). Significantly, three isoflavones exhibited a slight increase, and their structures were confirmed as daidzein, glycitein, and genistein, based on their molecular ions at m/z 253.1, 283.0, and 269.1 using the negative mode of HPLC-DAD-ESI/MS. The remaining compositions showed slight variations. The effects against 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radicals in healthy seeds were stronger than the diseased soybeans, depending upon the isoflavone level. Our results may be useful in evaluating the relationship between composition and antioxidant activity as a result of changes caused by soybean fungal pathogens.

Enzyme encapsulation in magnetic chitosan-Fe3O4 microparticles.

Two simple procedures for the preparation of magnetic chitosan enzyme microparticles have been investigated and used for the immobilisation of endophytic fungus Cercospora kikuchii lipase as model enzyme. In the first case, lipase was entrapped in Fe3O4-chitosan microparticles by cross-linking method, while in the second case magnetic immobilised derivatives were produced using spray drying. Immobilised enzymes showed high enzyme activity retention and stability during storage without significant loss of activity. Glutaraldehyde Fe3O4-chitosan powders presented a higher lipase activity retention and storage stability than the others preparations. However, the immobilised derivatives produced by cross-linking showed higher enzyme activity after reuse cycles. The results proved that the magnetic Fe3O4-chitosan microparticles are an effective support for the enzyme immobilisation since the immobilised lipase showed best properties than the free form.

Immobilization of Lipases Produced by the Endophytic Fungus Cercospora kikuchii on Chitosan Microparticles

This work studied the immobilization of Cercospora kikuchii lipases on chitosan microparticles by chemical attachment on chitosan acetate microparticles activated by glutaraldehyde (CAM) added before or after the enzyme and physical adsorption on highly deacetylated chitosan hydrochloride microparticles (CHM). Lipases covalently immobilized on pre-activated CAM showed better performance retaining 88.4% of the enzymatic activity, with 68.2% of immobilization efficiency (IE). The immobilized enzyme retained an activity of about 53.5 % after five reuses, using p-NPP as substrate. Physical adsorption of lipase onto highly deacetylated CHM showed 46.2 % of enzymatic activity and 28.6% of IE. This immobilized derivative did not lose activity up to 80 days of storage at 4°C, while lipases immobilized on pre-activated CAM maintained its activity up to 180 days at same conditions. Taken together the results indicate that chitosan microparticles provide an optimal microenvironment for the immobilized enzyme to maintain good activity and stability.

Detección de variabilidad genética en aislamientos de Cercospora kikuchii contaminantes de un mismo sembradío de soja / Detection of genetic variability in Cercospora kikuchii isolates from a single soybean field

El conocimiento de la epidemiología y la estructura poblacional de Cercospora kikuchii está poco desarrollado y no se han comunicado estudios al respecto en la Argentina. El objetivo de este trabajo fue seleccionar oligonucleótidos que permitan detectar variabilidad genética en aislamientos de C. kikuchii obtenidos a partir de soja proveniente de un mismo sembradío, mediante la aplicación de RAPD. Se trabajó con 6 aislamientos de C. kikuchii, 5 de ellos se obtuvieron a partir de trozos de tejido enfermo y el restante provenía de una colección de cultivos. De los 7 oligonucleótidos empleados, 5 resultaron útiles para el estudio poblacional de los aislamientos de C. kikuchii.

Current knowledge about epidemiology and population structure of Cercospora kikuchii is little developed and no studies regarding this subject have been reported in Argentina. The aim of this work was to select primers to study genetic variability in C. kikuchii isolated from the same soybean field using RAPD (Random Amplified Polymorphism DNA). RAPD was applied to the DNA of 5 C. kikuchii, isolated from diseased tissue of the soybean in the same field, another isolate, from a strain collection. Out of seven primers, five of them proved to be useful to study the population of C. kikuchii isolates.