Differential Carbon Catabolite Repression and Hemicellulolytic Ability among Pathotypes of Colletotrichum lindemuthianum against Natural Plant Substrates.

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops.

Selection of stable reference genes for qPCR expression of Colletotrichum lindemuthianum, the bean anthracnose pathogen.

Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), ß-tub (ß-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and ß-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.

Race Structure and Molecular Diversity of Colletotrichum lindemuthianum of Common Bean in Zambia.

Anthracnose, caused by the fungus Colletotrichum lindemuthianum, is a major disease of common bean (Phaseolus vulgaris L.) worldwide. C. lindemuthianum is genetically highly variable, and understanding the pathogen's diversity and distribution is a key step in developing common bean varieties with durable anthracnose resistance. The objectives of this study were to (i) characterize the race structure of C. lindemuthianum in Zambia and (ii) assess the molecular diversity of C. lindemuthianum in Zambia. A field survey was conducted in 20 bean-growing districts in Zambia to collect anthracnose symptomatic bean plants. A total of 103 C. lindemuthianum isolates were collected and characterized based on their reactions on 12 common bean race differential cultivars. RAM and ERIC-BOX DNA markers were used to assess molecular diversity of 60 isolates. A total of 58 races were characterized from the 103 isolates. Race 5 was the least virulent, and race 1631 was the most virulent based on their reaction on the 12 race differential cultivars. Race 19 had the highest recovery frequency (11%) and was the most extensively dispersed among the 22 bean-growing districts from where the isolates were collected. Only six races had previously been reported in Zambia, and 52 races were identified as new races reported for the first time in Zambia. Two races were virulent only on Andean cultivars, 11 races were virulent only on Middle American cultivars, and 45 races were virulent on both Andean and Middle American cultivars. No individual isolate showed pathogenicity on all the differential cultivars, and no isolate overcame the Co-4, Co-5, and Co-7 resistance gene pyramid that naturally exists in G2333. Phylogenetic analysis categorized the 60 isolates in six major clusters and six subclusters. The 60 isolates showed high genetic heterogeneity among and within a race of the same virulence. The study has revealed the existence of both Andean and Middle American races and extensive molecular diversity of C. lindemuthianum in Zambia. The knowledge on the race structure of C. lindemuthianum that this study has provided will be valuable for making breeding decisions on the host plant resistance genes required for developing common bean varieties with durable resistance to anthracnose in Zambia.

Genetic mapping of the Andean anthracnose resistance gene present in the common bean cultivar BRSMG Realce.

The rajado seeded Andean bean (Phaseolus vulgaris L.) cultivar BRSMG Realce (striped seed coat) developed by Embrapa expressed a high level of anthracnose resistance, caused by Colletotrichum lindemuthianum, in field and greenhouse screenings. The main goal of this study was to evaluate the inheritance of anthracnose resistance in BRSMG Realce, map the resistance locus or major gene cluster previously named as Co-Realce, identify resistance-related positional genes, and analyze potential markers linked to the resistance allele. F2 plants derived from the cross BRSMG Realce × BRS FC104 (Mesoamerican) and from the cross BRSMG Realce × BRS Notável (Mesoamerican) were inoculated with the C. lindemuthianum races 475 and 81, respectively. The BRSMG Realce × BRS FC104 F2 population was also genotyped using the DArTseq technology. Crosses between BRSMG Realce and BAT 93 (Mesoamerican) were also conducted and resulting F2 plants were inoculated with the C. lindemuthianum races 65 and 1609, individually. The results shown that anthracnose resistance in BRSMG Realce is controlled by a single locus with complete dominance. A genetic map including 1,118 SNP markers was built and shown 78% of the markers mapped at a distances less than 5.0 cM, with a total genetic length of 4,473.4 cM. A major locus (Co-Realce) explaining 54.6% of the phenotypic variation of symptoms caused by the race 475 was identified in Pv04, flanked by the markers snp1327 and snp12782 and 4.48 cM apart each other. These SNPs are useful for marker-assisted selection, due to an estimated selection efficiency of 99.2%. The identified resistance allele segregates independently of the resistance allele Co-33 (Pv04) present in BAT 93. The mapped genomic region with 704,867 bp comprising 63 putative genes, 44 of which were related to the pathogen-host interaction. Based on all these results and evidence, anthracnose resistance in BRSMG Realce should be considered as monogenic, useful for breeding purpose. It is proposed that locus Co-Realce is unique and be provisionally designated as CoPv04R until be officially nominated in accordance with the rules established by the Bean Improvement Cooperative Genetics Committee.

Genome-Wide Transcriptomic Analysis of the Effects of Infection with the Hemibiotrophic Fungus Colletotrichum lindemuthianum on Common Bean.

Bean anthracnose caused by the hemibiotrophic fungus Colletotrichum lindemuthianum is one of the most important diseases of common bean (Phaseolus vulgaris) in the world. In the present study, the whole transcriptome of common bean infected with C. lindemuthianum during compatible and incompatible interactions was characterized at 48 and 72 hpi, corresponding to the biotrophy phase of the infection cycle. Our results highlight the prominent role of pathogenesis-related (PR) genes from the PR10/Bet vI family as well as a complex interplay of different plant hormone pathways including Ethylene, Salicylic acid (SA) and Jasmonic acid pathways. Gene Ontology enrichment analysis reveals that infected common bean seedlings responded by down-regulation of photosynthesis, ubiquitination-mediated proteolysis and cell wall modifications. In infected common bean, SA biosynthesis seems to be based on the PAL pathway instead of the ICS pathway, contrarily to what is described in Arabidopsis. Interestingly, ~30 NLR were up-regulated in both contexts. Overall, our results suggest that the difference between the compatible and incompatible reaction is more a question of timing and strength, than a massive difference in differentially expressed genes between these two contexts. Finally, we used RT-qPCR to validate the expression patterns of several genes, and the results showed an excellent agreement with deep sequencing.

Phylogeny and pathogenicity of Colletotrichum lindemuthianum causing anthracnose of Phaseolus vulgaris cv. Bhaderwah-Rajmash from northern Himalayas, India.

With an annual loss of up to 100%, anthracnose caused by Colletotrichum is one of the most devastating diseases of common beans (Phaseolus vulgaris L.). Due to few distinctive morphological characters, Colletotrichum species are frequently misidentified. In India, several Colletotrichum species have been reported as pathogens of Phaseolus species, but none had previously been validated by means of molecular tools. In this study, we studied Colletotrichum strains from common beans cv. Bhaderwah-Rajmash from the northern Himalayas of India based on both morphological and DNA sequence data of six loci, namely ITS, gapdh, chs-1, his3, act, tub2. The strains were identified as C. lindemuthianum that belongs to the C. orbiculare species complex. Representative C. lindemuthianum strains tested on Phaseolus vulgaris cv. Bhaderwah-Rajmash were pathogenic and exhibited variation in symptomology and disease progression. By identifying the causal agent, we provided substantial information to develop the best control strategies for anthracnose of Phaseolus vulgaris from the northern Himalayas of India. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03216-0.

Optimizing the Agrobacterium tumefaciens-mediated transformation conditions in Colletotrichum lindemuthianum: a step forward to unravel the functions of pathogenicity arsenals.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 106 ml-1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail.

Characterization of Colletotrichum lindemuthianum Races in Zambia and Evaluation of the CIAT Phaseolus Core Collection for Resistance to Anthracnose.

Colletotrichum lindemuthianum, the causal pathogen of common bean (Phaseolus vulgaris) anthracnose, is highly variable. Therefore, understanding its race structure and identification of new sources of resistance is necessary for the development of varieties with durable resistance. The objectives of this study were (i) to characterize isolates of C. lindemuthianum collected from three major bean-growing regions in Zambia and (ii) evaluate the International Center for Tropical Agriculture (CIAT) Phaseolus core collection for resistance to C. lindemuthianum races 37, 73, and 566 and a blend of 20 races. Isolates collected from three major bean-growing districts in Zambia, namely Mporokoso, Mpika, and Mbala, were characterized as races 37, 73, and 566, respectively. A subset of the CIAT core collection comprising 885 accessions of common bean, 13 accessions of scarlet runner bean (Phaseolus coccineus), and 11 accessions of year bean (Phaseolus dumosus) was evaluated for resistance to races 37, 73, and 566 and a blend of 20 races in a greenhouse at the University of Zambia, Lusaka, Zambia. Totals of 72, 66, 48, and 9% of P. vulgaris accessions evaluated were highly resistant to races 37, 73, and 566 and a blend of 20 races, respectively. Also, accessions of P. coccineus and P. dumosus highly resistant to races 37, 73, and 566 were identified. Only eight of the 331 P. vulgaris accessions were highly resistant to all three individual races (37, 73, and 566) and to a blend of 20 races. These eight accessions constitute a valuable breeding resource for developing varieties with durable resistance to C. lindemuthianum.

Genome-Wide Identification of Key Components of RNA Silencing in Two Phaseolus vulgaris Genotypes of Contrasting Origin and Their Expression Analyses in Response to Fungal Infection.

RNA silencing serves key roles in a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity. Dicer, Argonaute (AGO), double-stranded RNA binding (DRB) proteins, RNA-dependent RNA polymerase (RDR), and DNA-dependent RNA polymerases known as Pol IV and Pol V form core components to trigger RNA silencing. Common bean (Phaseolus vulgaris) is an important staple crop worldwide. In this study, we aimed to unravel the components of the RNA-guided silencing pathway in this non-model plant, taking advantage of the availability of two genome assemblies of Andean and Meso-American origin. We identified six PvDCLs, thirteen PvAGOs, 10 PvDRBs, 5 PvRDRs, in both genotypes, suggesting no recent gene amplification or deletion after the gene pool separation. In addition, we identified one PvNRPD1 and one PvNRPE1 encoding the largest subunits of Pol IV and Pol V, respectively. These genes were categorized into subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization, and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different organs using online data and quantitative RT-PCR after pathogen infection. Several of the candidate genes were up-regulated after infection with the fungus Colletotrichum lindemuthianum.

Different loci control resistance to different isolates of the same race of Colletotrichum lindemuthianum in common bean.

KEY MESSAGE: Linkage and genome-wide association analyses using high-throughput SNP genotyping revealed different loci controlling resistance to different isolates of race 65 of Colletotrichum lindemuthianum in common bean. Development of varieties with durable resistance to anthracnose is a major challenge in common bean breeding programs because of the extensive virulence diversity of Colletotrichum lindemuthianum fungus. We used linkage and genome-wide association analyses to tap the genomic regions associated with resistance to different isolates of race 65. Linkage mapping was done using an F2 population derived from the cross between the Mesoamerican common beans BRS Estilo x Ouro Vermelho, inoculated with two different isolates of race 65. Association genetics relied on a diversity common bean panel containing 189 common bean accessions inoculated with five different isolates of race 65 as an attempt to validate the linkage analysis findings and, eventually, identify other genomic regions associated with resistance to race 65. The F2 population and diversity panel were genotyped with the BARCBean6K_3 Illumina BeadChip containing 5398 SNP markers. Both linkage and genome-wide association analyses identified different loci controlling resistance to different isolates of race 65 on linkage group Pv04. Genome-wide association analysis also detected loci on Pv05, Pv10 and Pv11 associated with resistance to race 65. These findings indicate that resistance to race 65 can be overcome by the virulence diversity among different isolates of the same race and could lead to the loss of resistance after cultivar release. We identified 25 resistant common bean cultivars to all five isolates of race 65 in the diversity panel. The accessions should be useful to develop cultivars combining different resistance genes that favor durable resistance to anthracnose in common bean.

Dry Bean and Anthracnose Development From Seeds With Varying Symptom Severity.

Anthracnose, caused by the fungal pathogen Colletotrichum lindemuthianum, is a damaging seed-transmitted disease of dry beans that causes reduced seed quality and yield. Seed-to-seedling transmission of C. lindemuthianum has been documented as high as 15% in asymptomatic seeds under greenhouse conditions. Increasing pathogen colonization in seeds has been correlated with increasing anthracnose seed symptoms via quantitative PCR (qPCR), but stem colonization has not been quantified. Previous studies also have characterized seed yield and quality losses caused by planting C. lindemuthianum-infected seeds, but none evaluated the effect of growing asymptomatic seeds on disease and plant development under field conditions. A real-time qPCR assay was developed in this study and used to detect C. lindemuthianum in the stems of seedlings as early as 15 days after planting. Field trials measured the seed-to-seedling transmission of C. lindemuthianum across levels of anthracnose symptoms in seeds ranging from healthy to severely discolored. Results from these two field trials indicated that emergence and yield decreased and foliar symptoms, pathogen detection, and incidence of symptoms on progeny seeds increased as the severity of infection in planted seeds increased. In both years, planting asymptomatic seeds resulted in higher anthracnose severity than planting healthy seeds. Yield, seed weight, and incidence of symptoms on progeny seeds were not higher in asymptomatic seeds than in healthy seeds in 2014, when moderate disease pressure was observed. However, these factors were significantly different in 2015, when anthracnose severity was driven up to 75% by conducive weather conditions. This serves as a strong warning to growers that planting seed grown in a field where anthracnose was present, even if those seeds are asymptomatic, can result in yield and quality losses. Planting certified dry bean seed is always recommended.

The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L.

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.

Genetics and mapping of a new anthracnose resistance locus in Andean common bean Paloma.

BACKGROUND: The Andean cultivar Paloma is resistant to Mesoamerican and Andean races of Colletotrichum lindemuthianum, the fungal pathogen that causes the destructive anthracnose disease in common bean. Remarkably, Paloma is resistant to Mesoamerican races 2047 and 3481, which are among the most virulent races of the anthracnose pathogen. Most genes conferring anthracnose resistance in common bean are overcome by these races. The genetic mapping and the relationship between the resistant Co-Pa gene of Paloma and previously characterized anthracnose resistance genes can be a great contribution for breeding programs. RESULTS: The inheritance of resistance studies for Paloma was performed in F2 population from the cross Paloma (resistant) × Cornell 49-242 (susceptible) inoculated with race 2047, and in F2 and F2:3 generations from the cross Paloma (resistant) × PI 207262 (susceptible) inoculated with race 3481. The results of these studies demonstrated that a single dominant gene confers the resistance in Paloma. Allelism tests performed with multiple races of C. lindemuthianum showed that the resistance gene in Paloma, provisionally named Co-Pa, is independent from the anthracnose resistance genes Co-1, Co-2, Co-3, Co-4, Co-5, Co-6, Co-12, Co-13, Co-14, Co-15 and Co-16. Bulk segregant analysis using the SNP chip BARCBean6K_3 positioned the approximate location of Co-Pa in the lower arm of chromosome Pv01. Further mapping analysis located the Co-Pa gene at a 390 kb region of Pv01 flanked by SNP markers SS82 and SS83 at a distance of 1.3 and 2.1 cM, respectively. CONCLUSIONS: The results presented here showed that Paloma cultivar has a new dominant gene conferring resistance to anthracnose, which is independent from those genes previously described. The linkage between the Co-Pa gene and the SS82 and SS83 SNP markers will be extremely important for marker-assisted introgression of the gene into elite cultivars in order to enhance resistance.

Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic Acid for Controlling Anthracnose in Common Bean Plants.

Kaurenoic acid derivatives were prepared and submitted to in vitro assays with the fungus Colletotrichum lindemuthianum, which causes anthracnose disease in the common bean. The most active substances were found to be methyl and p-bromobenzylesters, 7 and 9, respectively, of the hydrogenated kaurenoic acid, which presented a minimum inhibitory concentration (MIC) of 0.097 and 0.131 mM, respectively, while the commercial fungicide methyl thiophanate (MT) presented a MIC of 0.143 mM. Substances 7 (1.401 mM) and 9 (1.886 mM) reduced the severity of anthracnose in common bean to values statistically comparable to MT (2.044 mM). According to an in silico study, both compounds 7 and 9 are inhibitors of the ketosteroid isomerase (KSI) enzyme produced by other organisms, the amino acid sequence of which could be detected in fungal genomes. These substances appeared to act against C. lindemuthianum by inhibiting its KSI. Therefore, substances 7 and 9 are promising for the development of new fungicides.

Durabilidade da resistência induzida por ulvana e efeito da concentração de inóculo no controle da antracnose do feijão / Persistence of ulvan-induced resistance and effect of inoculum concentration in the control of bean anthracnose

O presente trabalho teve por objetivo avaliar a duração da resistência induzida, a eficiência do número e intervalo de aplicações de ulvana, bem como a influência de diferentes concentrações de inóculo no controle e na severidade da antracnose do feijão. Para tanto, foram realizados três experimentos independentes. Plantas de feijão (P. vulgaris, cv. Uirapuru) foram cultivadas em condições de casa-de-vegetação e, no primeiro experimento, foram pulverizadas com ulvana em uma única aplicação aos: nove dias antes da inoculação (dai) V3(i); seis dai V3(m); três dai V3(f); ou duas aplicações consecutivas realizadas aos nove e seis dai V3(i)/(m); nove e três dai V3(i)/(f) ou seis e três dai V3(m)/(f) e inoculadas em um mesmo momento. No segundo e terceiro experimentos, plantas de feijão foram tratadas duas vezes (seis e três dai) e inoculadas em diferentes momentos ou com diferentes concentrações de inóculo, respectivamente. A pulverização de ulvana reduziu a severidade da antracnose em cerca de 50%. Duas aplicações de ulvana foram mais eficientes em induzir respostas de defesa em feijão contra a antracnose que somente uma. A maior redução (96%) da doença ocorreu com pulverizações sucessivas em V3(m)/(f). O efeito de duas aplicações do polissacarídeo persistiu até nove dias após o tratamento. A redução na severidade da antracnose foi maior quando foram utilizadas concentrações intermediárias de inóculo (105 e 106 conídios por mL).

The present work aimed to evaluate the persistence of the induced resistance, the efficiency of the number and the interval of ulvan applications and the influence of different inoculum concentrations in the control of bean anthracnose. Three independent experiments were carried out. Bean plants (P. vulgaris, cv. Uirapuru) were cultivated under green house conditions and, in the first experiment, they were treated with ulvan once at nine days before inoculation (dbi) V3(i); six dbi V3(m) or three dbi V3(f) or they were treated twice at nine and six dbi V3(i)/(m); nine and three V3(i)/(f) or six and three (dbi) V3(m)/(f), the they were inoculated at the same time. In the second and third experiments, bean plants were treated twice (six and three dbi) then inoculated in different moments or with different inoculums concentrations, respectively. The application of ulvan reduced the anthracnose severity by 50%. Two ulvan applications were more efficient in inducing defense responses in bean plants than a single application. The highest disease reduction (96%) was achieved by spraying ulvan twice at V3(m)/(f). The effect of two applications persists until nine days after the treatment. The reduction on the anthracnose severity was higher when intermediate (105 e 106 conidia per mL) inoculum concentrations were used.

Vegetative compatibility and heterokaryon formation between different isolates of Colletotrichum lindemuthianum by using the nit mutant system

Colletotrichum lindemuthianum, the causative agent of bean anthracnose, is one of the most common pathogens leading to expressive damage to plants beyond presenting noticeable variability. The knowledge on vegetative compatibility groups (VCGs) is of particular interest in asexual fungi as they subdivide the population in groups that can exchange genetic information via heterokaryosis and the parasexual cycle. Among the techniques used in studies about vegetative compatibility groups, the obtainment of nit mutants is apparent. This paper is aimed at obtaining heterokaryons between different isolates of C. lindemuthianum, grouping them in VCGs and evaluating their genetic variability by using the nit mutants system. Nit mutants were obtained from 20 single spore isolates. The mutants were phenotypically classified and paired for complementation and formation of heterokaryons so as to group them in VCGs. Seventeen mutants from the different phenotypic-rates were recovered: nit1, nit2, nit3 and nitM. At the same time, 10 mutants were selected for pairing and division of the anastomosis groups. Nine heterokaryons were obtained and the isolates were divided into 9 vegetative compatibility groups. In the combinations for the formation of anastomosis, 31 compatible combinations and 24 incompatible combinations were observed. It was concluded that the methodology used to select nit mutants in C. lindemuthianum made it possible to determine the vegetative compatibility groups and that such a technique was adequate to prove genetic variability.

Solid state fermentation production of chitin deacetylase by Colletotrichum lindemuthianum ATCC 56676 using different substrates.

Production of extracellular chitin deacetylase by Colletotrichum lindemuthianum ATCC 56676 under solid substrate fermentation was studied. The suitability of shrimp shell chitin waste (SSCW) and commercial wheat bran (CWB) was evaluated for maximal enzyme production. CWB medium (pH 6.4 ± 0.2) supplemented with chitosan favoured maximal chitin deacetylase yield of 460.4 ± 14.7 unit/g initial dry substrate (U/g IDS) at 96 h as compared to maximal yield of 392.0 ± 6.4 U/g IDS at 192 h in SSCW medium (pH 8.7 ± 0.2) at 25 °C incubation temperature and 60% (w/w) initial moisture content of medium. Along with chitin deacetylase, C. lindemuthianum ATCC 56676 produced maximum endo-chitinase (0.28 ± 0.03 U/g IDS at 144 h) and ß-N-acetylhexosaminidase (0.79 ± 0.009 U/g IDS at 192 h) in CWB medium and 0.49 ± 0.05 U/g IDS of endo-chitinase at 264 h and 0.38 ± 0.04 U/g IDS of ß-N-acetylhexosaminidase at 96 h of incubation in SSCW medium. SEM studies indicated the difference in the morphology of mycelia and hyphae of C. lindemuthianum ATCC 56676 when grown on different solid substrates. Production of chitin deacetylase by SSF is being reported for the first time.

Vegetative Compatibility and Heterokaryon Formation between Different Isolates of Colletotrichum Lindemuthianum by using the nit Mutant System.

Colletotrichum lindemuthianum, the causative agent of bean anthracnose, is one of the most common pathogens leading to expressive damage to plants beyond presenting noticeable variability. The knowledge on vegetative compatibility groups (VCGs) is of particular interest in asexual fungi as they subdivide the population in groups that can exchange genetic information via heterokaryosis and the parasexual cycle. Among the techniques used in studies about vegetative compatibility groups, the obtainment of nit mutants is apparent. This paper is aimed at obtaining heterokaryons between different isolates of C. lindemuthianum, grouping them in VCGs and evaluating their genetic variability by using the nit mutants system. Nit mutants were obtained from 20 single spore isolates. The mutants were phenotypically classified and paired for complementation and formation of heterokaryons so as to group them in VCGs. Seventeen mutants from the different phenotypic-rates were recovered: nit1, nit2, nit3 and nitM. At the same time, 10 mutants were selected for pairing and division of the anastomosis groups. Nine heterokaryons were obtained and the isolates were divided into 9 vegetative compatibility groups. In the combinations for the formation of anastomosis, 31 compatible combinations and 24 incompatible combinations were observed. It was concluded that the methodology used to select nit mutants in C. lindemuthianum made it possible to determine the vegetative compatibility groups and that such a technique was adequate to prove genetic variability.

Seleção de linhagens de feijoeiro com tipo de grão carioca e com os alelos co-4 e co-5 de resistência à antracnose / Selection of common bean strains with carioca grain type, and with the alleles co-4 and co-5 for anthracnose resistance

Objetivou-se, neste trabalho, identificar linhagens de feijão que reúnam, além da resistência à antracnose, alta produtividade de grãos do tipo carioca e resistência à mancha angular. Foram utilizadas 194 linhagens F5:6 extraídas de sete famílias segregantes, selecionadas do cruzamento entre os genitores H147 e B1. A linhagem H147 possui grãos tipo carioca, portadora do alelo Co-5, que confere resistência a várias raças de Colletotrichum lindemuthianum. A linhagem B1 também possui grãos tipo carioca e é portadora do alelo Co-4, que confere resistência a outro grupo de raças do mesmo patógeno. As linhagens foram avaliadas na safra das águas 2005/2006, em Lavras, com a cultivar Talismã e H147 como testemunhas, com base na produtividade e tipo de grãos. Foram selecionadas 99 linhagens, as quais foram avaliadas na safra da seca/2006, juntamente com a testemunha Talismã, com base na produtividade, tipo de grão e resistência à mancha angular. Dessas 99 linhagens, foram selecionadas 24, as quais foram avaliadas na safra de inverno/2006 em Lavras e Lambari, com base no tipo de grão e produtividade. Essas 24 linhagens foram inoculadas com a raça 321 de C. lindemuthianum, que quebra a resistência conferida pelo alelo Co-4, mas não o Co-5. Para verificar a presença do alelo Co4 foi utilizado um marcador SCAR que amplifica um fragmento de 950 pb por meio do primer SAS 13. Foi possível identificar 14 linhagens que possuem a pirâmide de alelos Co-4/Co-5 e entre elas, quatro destacaram-se em todos os caracteres avaliados.

The objective of the research was to identify bean strains that possess at the same time resistance to anthracnose, high grain yield of Carioca grain type and resistance to angular leaf spot. 194 strains F5:6 were taken from seven segregating families derived from the cross H147 x B1. The H147 line has Carioca grain type and Co-5 resistance allele to several races of C. lindemuthianum. The B1 line also has the Carioca grain type, and the Co-4 resistance allele against others races of the same pathogen. The strains were evaluated in the spring/summer (2005/2006) in the municipality of Lavras, in the Southern of the State of Minas Gerais, with the Talismã cultivar and H147 line as check, based mainly on grain type and grain yield. 99 strains, together with the Talismã check, were selected and evaluated based on grain yield, grain type and angular leaf spot resistance in the summer (2006) in the same place. From these 99 strains, 24 strains were selected and evaluated in the winter/spring (2006) in two places, Lavras and Lambari, based on the type of grain and productivity, and using the same check. Those 24 strains were also inoculated with the race 321 of C. lindemuthianum that break the resistance of the Co-4 allele, but not of the Co-5. The SCAR marker using the SAS13 primer was used to detect the presence of the Co-4 allele. Fourteen strains were identified that have the Co-4/Co-5 allele pyramid, and, among them, four stand out in all evaluated features.

Intensity of angular leaf spot and anthracnose on pods of common beans cultivated in three cropping systems / Intensidade da mancha-angular e da antracnose em vagens de feijão em três sistemas de cultivo

With the purpose to evaluate the intensity of angular leaf spot (ALS) and anthracnose (ANT) on pods, nine genotypes of common bean were planted in three cropping systems: monocrop (MC), monocrop grown on trellises (MCT), and intercrop with maize (ICM). In MC, beans were planted 0.5 m apart. Trelisses were set up with 1.8 m high bamboos and beans were sown 0.65 m apart. In ICM, beans were planted simultaneously with maize and in its rows. This cereal was sown 1.0 m apart with four plants per meter. Each cropping system was an independent trial installed close to each other. Climbing genotypes of beans most susceptible to ALS had less diseased pods in ICM than in both MC and MCT, but the less susceptible genotypes, regardless of their growth type, as well as the susceptible bush and semiclimbing genotypes, were similarly attacked by ALS in the three systems. ANT on pods of the susceptible bean cv. Pérola was less intense in MCT than in MC, and less intense in ICM than in MCT. ANT seed transmission was 11 percent, 9.1 percent, and 4.4 percent when seeds come from MC, MCT, and ICM, respectively.

Com o objetivo de avaliar a intensidade da mancha-angular (MA) e da antracnose (ANT) em vagens, nove genótipos de feijão foram plantados em três sistemas de plantio: monocultivo (MC), monocultivo com tutoragem artificial (MCT) e consórcio com milho (CCM). No MC, o feijão foi semeado no espaçamento entre fileiras de 0,50 m. Para o MCT, utilizaram-se varas de bambu formando "Vs" invertidos com 1,8 m de altura, nos quais foi mantido o espaçamento entre fileiras de 0,65 m na base. No CCM, o feijão foi plantado simultaneamente ao milho e em suas fileiras. Esse cereal foi semeado no espaçamento de 1,0 m, com quatro plantas por metro. Cada sistema de cultivo foi um ensaio independente, todos instalados na mesma área. Os genótipos trepadores mais suscetíveis à MA apresentaram menos vagens doentes no CCM que no MC e MCT, mas os genótipos menos atacados pela MA, independentemente do tipo de crescimento, como também os outros genótipos suscetíveis de porte anão e semitrepador, foram infectados de forma semelhante nos três sistemas de plantio. A ANT nas vagens do cv. Pérola foi menos intensa no MCT que no MC, e menos intensa no CCM que no MCT. A transmissão da ANT pela semente foi de 11 por cento, 9,1 por cento e 4,4 por cento quando as sementes foram colhidas no MC, MCT e CCM, respectivamente.