Fungus Colletotrichum Lindemuthianum Research Articles

<b>Classification of <i>Colletotrichum lindemuthianum</i> races in differential cultivars of common bean

Anthracnose caused by the fungus Colletotrichum lindemuthianum is one of the main diseases affecting the common bean ( Phaseolus vulgaris L.), and the pathogen is characterized by wide variability, with more than 50 physiological races identified in Brazil. Greater occurrences of races 65, 73, and 81 have been observed in Brazil along with the occurrence of pathogenic variability among isolates of a single race, destabilizing the resistance of commercial cultivars. Therefore, the aim of this study was to identify physiological races of C. lindemuthianum isolates collected in the states of Sao Paulo and Santa Catarina, Brazil and to test for variability among the isolates of race 65. The classification of 51 isolates resulted in the identification of 10 different physiological races: 4, 38, 55, 65, 73, 81, 83, 85, 321, and 351. Races 65 and 81 predominated, with frequencies of 37.25 and 35.29%, respectively. Regarding the isolates of race 65, wide physiological variability was evident, suggesting that a new differential set should be applied to detect the levels of variation among isolates of a single race of the pathogen.

Purification, biochemical characterization, and antimicrobial activity of a new lipid transfer protein from Coffea canephora seeds.

Coffee, an agronomical crop of great economic importance, is also among the most commonly traded commodities in worldwide markets. Antimicrobial peptides, which play a role in plant defense, have been identified and isolated particularly from seeds. We isolated and immunolocalized Cc-LTP2, a new lipid transfer protein (LTP) from Coffea canephora seeds. We report its antimicrobial activity against various phytopathogenic fungi of economic importance, and against the bacterium Xanthomonas euvesicatoria. Peptides from C. canephora seeds were initially extracted using acid buffer and subjected to ion-exchange and reverse-phase chromatographies. A purified peptide of approximately 9 kDa, which we named Cc-LTP2, was then subjected to amino acid sequencing. The analyses showed that it was similar to LTPs isolated from various plants. The tissue and subcellular localization of C. canephora LTPs indicated that they were located in cell walls and intracellular palisade parenchyma, mainly in large vacuoles. The results of immunohistochemistry and histochemistry superposed from C. canephora seed tissues showed that LTPs and lipid bodies are present in organelles, supporting the hypothesis that LTPs from seeds are involved in lipid mobilization during germination. Cc-LTP2 did inhibit the development of the phytopathogenic fungi Colletotrichum lindemuthianum, Colletotrichum gloeosporioides, Fusarium solani, Fusarium lateritium, and Colletotrichum sp, but did inhibit X. euvesicatoria. Cc-LTP2 also increased membrane permeability and induced endogenous production of reactive oxygen species in all the fungi tested.

The Co-4 locus on chromosome Pv08 contains a unique cluster of 18 COK-4 genes and is regulated by immune response in common bean.

The common bean locus Co - 4, traditionally referred to as an anthracnose-resistant gene, contains a cluster of predicted receptor-like kinases (COK-4 and CrRLK1-like), and at least two of these kinases are co-regulated with the plant's basal immunity. Genetic resistance to anthracnose, caused by the fungus Colletotrichum lindemuthianum (Sacc. and Magnus) Briosi and Cavara, is conferred by major loci throughout the Phaseolus vulgaris genome, named Co. The complex Co-4 locus was previously reported to have several copies of the COK-4 gene that is predicted to code for a receptor-like kinase (RLK). In general, plant RLKs are involved in pathogen perception and signal transduction; however, the molecular function of COK-4 remains elusive. Using newly identified molecular markers (PvTA25 and PvSNPCOK-4), the SAS13 marker, COK-4 sequences and phylogeny, and the recently released bean genome sequence, we determined the most probable boundaries of the Co-4 locus: a 325-Kbp region on chromosome Pv08. Out of the 49 predicted transcripts in that region, 24 encode for putative RLKs (including 18 COK-4 copies) with high similarity to members of the Catharanthus roseus RLK1-like (CrRLK1L) protein family from different plant species, including the well-described FERONIA (FER) and ANXUR. We also determined that two RLK-coding genes in the Co-4 locus (COK-4-3 and FER-like) are transcriptionally regulated when bean plants are challenged with the flg22 peptide, a commonly used elicitor of plant immunity, or the bacterium Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight. While COK-4-3 is activated during immune response, FER-like is downregulated suggesting that these genes could play a role in plant responses to biotic stress. These results highlight the importance of dissecting the regulation and molecular function of individual genes within each locus, traditionally referred to as resistance gene based on genetic segregation analysis.

Plant volatiles cause direct, induced and associational resistance in common bean to the fungal pathogen Colletotrichum lindemuthianum

Summary Plants that express resistance to herbivores emit volatile organic compounds (VOCs) that can trigger resistance responses in undamaged neighbours. Recent reports indicate that VOCs can also trigger the resistance to pathogens, an effect that might be due to different mechanisms: the priming of an induced expression of resistance genes in the receiver or direct inhibitory effects on microbial pathogens that cause a passive ‘associational’ resistance in the VOC‐exposed plant. We investigated whether VOCs emitted from a resistant common bean (Phaseolus vulgaris) cultivar enhance the resistance to the fungus Colletotrichum lindemuthianum in a susceptible cultivar and analysed whether specific VOCs are likely to directly affect the pathogen. We found that susceptible plants exposed to the headspace of resistance‐expressing plants over 6 h became phenotypically as resistant as the resistant cultivar. Several resistance marker genes (PATHOGENESIS‐RELATED [PR] 1, 2 and 4) were primed in VOC‐exposed susceptible plants. After challenging, these genes reached expression levels at least as high as in the resistant cultivar. Additionally, individual VOCs such as limonene, linalool, nonanal, methyl salicylate and methyl jasmonate at natural concentrations directly inhibited the germination of conidia as did also the headspace of a resistance‐expressing plant. This inhibition of conidial germination was dosage‐dependent and irreversible. Synthesis. We conclude that VOCs are involved in the resistance of bean to fungal pathogens. They can contribute to the direct resistance in the emitter itself, and resistance phenotypes of neighbouring receiver plants can result from induced as well as associational resistance. Plant VOCs play multiple roles in the resistance of plants to microbial pathogens.

Caracterização de Raças de Colletotrichum lindemuthianum no Estado do Paraná

Anthracnose is a plant disease caused by fungus Colletotrichum lindemuthianum and may attack the aerial parts of the bean plant. The pathogen may vary in virulence and continuous studies are relevant on its variability so that strains may be detected and resistant cultivars may be developed. Current analysis identifies the C. lindemuthianum strains in the state of Parana, Brazil, and updates knowledge on its variability. Isolations of plants from different regions of the state and the monospore of each isolate were obtained. A group of twelve differentiating varieties and the binary system was used for strain characterization. Monospores were ground in concentrations of 1.2x106 conidia/mL in the differentiating varieties, which were then placed in a acclimatized room. Assessment occurred on the seventh day and the susceptibility of the varieties provided the identification of ten strains, namely, 65, 73, 75, 83, 85, 89, 95, 457, 475 and 1.609. This is the first report of strain 1.609 in the state of Parana, Brazil. Its occurrence is important for the genetic improvement of the bean plant, since it infects the differentiating varieties TU and AB 136 which had been resistant to all strains in the state.

Aspectos microscópicos da interação feijoeiro-Colletotrichum lindemuthianum mediados pelo silício

A antracnose, causada pelo fungo Colletotrichum lindemuthianum, é uma das doenças mais destrutivas que afetam a cultura do feijoeiro. Com o objetivo de encontrar alternativas para o controle dessa doença, o presente trabalho investigou, em nível microscópico, o efeito do silício (Si) na resistência do feijoeiro à infecção por C. lindemuthianum. Plantas de feijoeiro (cv. Pérola) foram cultivadas em solução nutritiva contendo 0 (-Si) ou 2 mM (+Si) de Si e inoculadas no estádio de crescimento V4 com uma suspensão de conídios de C. lindemuthianum. A severidade da antracnose decresceu cerca de 52% nas folhas das plantas supridas com Si (4,4%) em relação às folhas das plantas não supridas (8,5%). Observações de folhas de feijoeiro das plantas não supridas com Si no microscópio eletrônico de varredura revelaram alterações morfológicas nas nervuras em contraste com as folhas de plantas supridas com Si. Utilizando-se a microanálise de raios-X, verificou-se maior concentração dos minerais enxofre, potássio e Si nas folhas das plantas supridas com Si. Em conclusão, o suprimento de Si em plantas de feijoeiro foi importante para reduzir os sintomas da antracnose.

Order of arrival shifts endophyte-pathogen interactions in bean from resistance induction to disease facilitation.

Endophytic fungi colonize plants without causing symptoms of disease and can enhance the resistance of their host to pathogens. We cultivated 53 fungal strains from wild lima bean (Phaseolus lunatus) and investigated their effects on pathogens using in vitro assays and experiments in planta. Most strains were annotated as Rhizopus, Fusarium, Penicillium, Cochliobolus, and Artomyces spp. by the sequence of their 18S rRNA gene. In vitro confrontation assays between endophytes and three pathogens (the bacteria Pseudomonas syringae pv. syringae and Enterobacter sp. strain FCB1, and the fungus Colletotrichum lindemuthianum) revealed strong and mainly symmetric reciprocal effects: endophyte and pathogen either mutually inhibited (mainly Enterobacter FCB1 and Colletotrichum) or facilitated (P. syringae) the growth of each other. In planta, the endophytes had a strong inhibitory effect on P. syringae when they colonized the plant before the bacterium, whereas infection was facilitated when P. syringae colonized the plant before the endophyte. Infection with Enterobacter FCB1 was facilitated when the bacterium colonized the plant before or on the same day with the endophyte, but not when the endophyte was present before the bacterium. The order of arrival determines whether fungal endophytes enhance plant resistance to bacterial pathogens or facilitate disease.

Fine mapping of Co-x, an anthracnose resistance gene to a highly virulent strain of Colletotrichum lindemuthianum in common bean.

The Co - x anthracnose R gene of common bean was fine-mapped into a 58 kb region at one end of chromosome 1, where no canonical NB-LRR-encoding genes are present in G19833 genome sequence. Anthracnose, caused by the phytopathogenic fungus Colletotrichum lindemuthianum, is one of the most damaging diseases of common bean, Phaseolus vulgaris. Various resistance (R) genes, named Co-, conferring race-specific resistance to different strains of C. lindemuthianum have been identified. The Andean cultivar JaloEEP558 was reported to carry Co-x on chromosome 1, conferring resistance to the highly virulent strain 100. To fine map Co-x, 181 recombinant inbred lines derived from the cross between JaloEEP558 and BAT93 were genotyped with polymerase chain reaction (PCR)-based markers developed using the genome sequence of the Andean genotype G19833. Analysis of RILs carrying key recombination events positioned Co-x at one end of chromosome 1 to a 58 kb region of the G19833 genome sequence. Annotation of this target region revealed eight genes: three phosphoinositide-specific phospholipases C (PI-PLC), one zinc finger protein and four kinases, suggesting that Co-x is not a classical nucleotide-binding leucine-rich encoding gene. In addition, we identified and characterized the seven members of common bean PI-PLC gene family distributed into two clusters located at the ends of chromosomes 1 and 8. Co-x is not a member of Co-1 allelic series since these two genes are separated by at least 190 kb. Comparative analysis between soybean and common bean revealed that the Co-x syntenic region, located at one end of Glycine max chromosome 18, carries Rhg1, a major QTL contributing to soybean cyst nematode resistance. The PCR-based markers generated in this study should be useful in marker-assisted selection for pyramiding Co-x with other R genes.

Genetic analysis of the response to eleven Colletotrichum lindemuthianum races in a RIL population of common bean (Phaseolus vulgaris L.).

BackgroundBean anthracnose is caused by the fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.- Scrib. Resistance to C. lindemuthianum in common bean (Phaseolus vulgaris L.) generally follows a qualitative mode of inheritance. The pathogen shows extensive pathogenic variation and up to 20 anthracnose resistance loci (named Co-), conferring resistance to specific races, have been described. Anthracnose resistance has generally been investigated by analyzing a limited number of isolates or races in segregating populations. In this work, we analyzed the response against eleven C. lindemuthianum races in a recombinant inbred line (RIL) common bean population derived from the cross Xana × Cornell 49242 in which a saturated linkage map was previously developed.ResultsA systematic genetic analysis was carried out to dissect the complex resistance segregations observed, which included contingency analyses, subpopulations and genetic mapping. Twenty two resistance genes were identified, some with a complementary mode of action. The Cornell 49242 genotype carries a complex cluster of resistance genes at the end of linkage group (LG) Pv11 corresponding to the previously described anthracnose resistance cluster Co-2. In this position, specific resistance genes to races 3, 6, 7, 19, 38, 39, 65, 357, 449 and 453 were identified, with one of them showing a complementary mode of action. In addition, Cornell 49242 had an independent gene on LG Pv09 showing a complementary mode of action for resistance to race 453. Resistance genes in genotype Xana were located on three regions involving LGs Pv01, Pv02 and Pv04. All resistance genes identified in Xana showed a complementary mode of action, except for two controlling resistance to races 65 and 73 located on LG Pv01, in the position of the previously described anthracnose resistance cluster Co-1.ConclusionsResults shown herein reveal a complex and specific interaction between bean and fungus genotypes leading to anthracnose resistance. Organization of specific resistance genes in clusters including resistance genes with different modes of action (dominant and complementary genes) was also confirmed. Finally, new locations for anthracnose resistance genes were identified in LG Pv09.

Influence of Contaminated Crop Remains and Seed Health Quality on the Intensity of Bean Anthracnose

Bean anthracnose, caused by the fungus Colletotrichum lindemuthianum, is a major seed borne disease of beans, interfering with the progress of the disease timeline. Work involving the study of cultural remains are scarce. The effects of different incidences of that pathogen in seeds in combination with cultivating areas with and without contaminated crop remains were evaluated on the anthracnose incidence and severity and on yield. In the trial set up in December 2009 the cultivation of bean in area with contaminated crop remains had greater progress for both incidence and severity of the disease compared with the area without crop remains. There was interaction between infection levels in seeds with or without contaminated crop remains in both growing areas. In the trial set up in April 2010 progress of anthracnose was similar in both growing areas. Grain yield was higher in the area without crop remains in both trials. The results of this work showed that the behavior of anthracnose under field conditions differ among areas studied (with and without crop residues) and also due to the environmental conditions of a planting to another.

Beginning to understand the role of sugar carriers in Colletotrichum lindemuthianum: the function of the gene mfs1

Fungi of the Colletotrichum genus are among the most prominent phytopathogens that cause diseases with a considerable economic impact, such as anthracnose. The hemibiotrophic fungus Colletotrichum lindemuthianum (teleomorph Glomerella cingulata f. sp. phaseoli) is the causal agent of the anthracnose of the common bean; and similarly to other phytopathogens, it uses multiple strategies to gain access to different carbon sources from its host. In this study, we examine mfs1, a newly identified C. lindemuthianum hexose transporter. The mfs1 gene is expressed only during the necrotrophic phase of the fungus' interaction within the plant and allows it to utilize the available sugars during this phase. The deletion of mfs1 gene resulted in differential growth of the fungus in a medium that contained glucose, mannose or fructose as the only carbon source. This study is the first to describe a hexose transporter in the hemibiotrophic pathogen C. lindemuthianum and to demonstrate the central role of this protein in capturing carbon sources during the necrotrophic development of the plant/pathogen interaction.

Morphological and molecular screening of French bean (Phaseolus vulgaris L.) germplasm using SCAR markers for Colletotrichum lindemuthianum (Sacc. and Magn.) Scrib. causing anthracnose resistance

This study was carried out to screen Phaseolus vulgaris L. germplasm accessions for anthracnose resistance genes to the fungus Colletotrichum lindemuthianum. (Sacc. and Magn.) Scrib. This fungus is made up of many pathogenic races which poses a challenge in developing resistant plant varieties; however, screening for and selection of resistant plant sources plays an important role in developing resistant plant lines. This screening work involved examining 69 accessions consisting of two resistant lines (D-line, L-line) and two susceptible varieties (Kanchana and Jwala). Fourteen SCAR primers specific to anthracnose disease resistance in the French bean were used. Of these 14 SCAR primers, 5 of them, SAS13, SF10, SC08, SZ04 and SBB14, produced amplification with good monomorphic bands.

Angiotensin‐converting enzyme‐inhibitory activity in protein hydrolysates from normal and anthracnose disease‐damaged Phaseolus vulgaris seeds

Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above-ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin-converting enzyme (ACE-I)-inhibitory activity. Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE-I-inhibitory activity (IC(50) 0.0191 mg protein mL(-1)) and were very similar to those from control beans in terms of ACE-I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product. The present results suggest the use of spoiled bean seeds, e.g. anthracnose-damaged beans, as an alternative for the isolation of ACE-I-inhibitory peptides to be further introduced as active ingredients in functional foods.

Changes in spatial and temporal gene expression during incompatible interaction between common bean and anthracnose pathogen

Common bean, one of the most important legumes for human consumption, may have drastic reduction in yield due to anthracnose, a disease caused by the fungus Colletotrichum lindemuthianum. Rapid induction of the plant defense mechanisms is essential to establish an incompatible interaction with this pathogenic fungus. In this study, we evaluated spatial (leaves, epicotyls and hypocotyls) and temporal (24, 48, 72 and 96 hours after inoculation [HAI]) relative expression (RE) of 12 defense-related transcripts selected from previously developed ESTs libraries, during incompatible interaction between the resistant common bean genotype SEL 1308 and the avirulent anthracnose pathogen race 73, using real time quantitative RT-PCR (RT-qPCR) analysis. All selected transcripts, including the ones coding for pathogenesis-related (PR) proteins (PR1a, PR1b, PR2, and PR16a and PR16b) were differentially regulated upon pathogen inoculation. The expression levels of these transcripts were dependent on the tissue and time post inoculation. This study contributes to a better understanding of the kinetics of induced defenses against a fungal pathogen of common bean and may be used as a base line to study defenses against a broad range of pathogens including bacteria as well as non-host resistance.

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Development of new molecular markers for the Colletotrichum genus using RetroCl1 sequences

A nonautonomous element of 624 bp, called RetroCl1 (Retroelement Colletotrichum lindemuthianum 1), was identified in the plant pathogenic fungus Colletotrichum lindemuthianum. RetroCl1 contains terminal direct repeats (223 bp) that are surrounded by CTAGT sequences. It has a short internal domain of 178 bp and shows characteristics of terminal-repeat retrotransposon in miniature (TRIM) family. We used RetroCl1 sequence to develop molecular markers for the Colletotrichum genus. IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) markers were used to analyze the genetic diversity of C. lindemuthianum. Fifty-four isolates belonging to different races were used. A total of 45 loci were amplified. The Nei index showed significant differences among the populations divided according to race, indicating that they are structured according to pathotype. No clear correlation between IRAP and REMAP markers with pathogenic characterization was found. C. lindemuthianum has high genetic diversity, and the analysis of molecular variance showed that 51% of variability is found among the populations of different races. The markers were also tested in different Colletotrichum species. In every case, multiple bands were amplified, indicating that these markers can be successfully used in different species belonging to the Colletotrichum genus.

Genetic Analysis of the Resistance to Eight Anthracnose Races in the Common Bean Differential Cultivar Kaboon

Resistance to the eight races (3, 7, 19, 31, 81, 449, 453, and 1545) of the pathogenic fungus Colletotrichum lindemuthianum (anthracnose) was evaluated in F(3) families derived from the cross between the anthracnose differential bean cultivars Kaboon and Michelite. Molecular marker analyses were carried out in the F(2) individuals in order to map and characterize the anthracnose resistance genes or gene clusters present in Kaboon. The analysis of the combined segregations indicates that the resistance present in Kaboon against these eight anthracnose races is determined by 13 different race-specific genes grouped in three clusters. One of these clusters, corresponding to locus Co-1 in linkage group (LG) 1, carries two dominant genes conferring specific resistance to races 81 and 1545, respectively, and a gene necessary (dominant complementary gene) for the specific resistance to race 31. A second cluster, corresponding to locus Co-3/9 in LG 4, carries six dominant genes conferring specific resistance to races 3, 7, 19, 449, 453, and 1545, respectively, and the second dominant complementary gene for the specific resistance to race 31. A third cluster of unknown location carries three dominant genes conferring specific resistance to races 449, 453, and 1545, respectively. This is the first time that two anthracnose resistance genes with a complementary mode of action have been mapped in common bean and their relationship with previously known Co- resistance genes established.

Use of plant extracts in the control of common bean anthracnose

Three hundred (300) methanol extracts of barks, leaves, flowers and stems of 200 plant species from Alto Rio Grande and Vale do São Francisco regions, State of Minas Gerais, Brazil, were prepared and submitted to an in vitro growth test with the fungus Colletotrichum lindemuthianum, causal agent of anthracnose in common bean. Extracts from 13 plant species ( Astronium fraxinifolium, Inga marginata, Malva sylvestris, Matayba elaeagnoides, Miconia argyrophylla, Myrcia fallax, Ocimum gratissimum, Origanum vulgare, Rollinia emarginata, Siparuna arianeae, Styrax pohlii, Tabebuia serratifolia and Trichilia pallida) presented antifungal activity and were used in other in vitro assays. Extracts from M. argyrophylla, M. fallax, O. vulgare, S. arianeae and S. pohlii were the most promising for the inhibition of the mycelial growth while the extracts of M. argyrophylla, M. elaeagnoides and O. gratissimum presented the best results for the inhibition of conidial germination. Under greenhouse conditions, the extracts of M. argyrophylla and O. vulgare caused the greatest reductions (41.82% and 37.65%, respectively) in disease severity when a local effect assay was carried out. In the systemic effect assay, also in a greenhouse, the most promising extracts were those from I. marginata, M. argyrophylla, M. fallax, M. sylvestris, O. gratissimum, O. vulgare and S. arianeae, which reduced the severity of the anthracnose to values below 35% of the observed for the control. Therefore, future studies with these plant species should be carried out to develop new products to control the common bean anthracnose.

Three highly similar formate dehydrogenase genes located in the vicinity of the B4 resistance gene cluster are differentially expressed under biotic and abiotic stresses in Phaseolus vulgaris

In higher plants, formate dehydrogenase (FDH, EC1.2.1.2.) catalyzes the NAD-linked oxidation of formate to CO(2), and FDH transcript accumulation has been reported after various abiotic stresses. By sequencing a Phaseolus vulgaris BAC clone encompassing a CC-NBS-LRR gene rich region of the B4 resistance gene cluster, we identified three FDH-encoding genes. FDH is present as a single copy gene in the Arabidopsis thaliana genome, and public database searches confirm that FDH is a low copy gene in plant genomes, since only 33 FDH homologs were identified from 27 plant species. Three independent prediction programs (Predotar, TargetP and Mitoprot) used on this large subset of 33 plant FDHs, revealed that mitochondrial localization of FDH might be the rule in higher plants. A phylogenetic analysis suggests a scenario of local FDH gene duplication in an ancestor of the Phaseoleae followed by another more recent duplication event after bean/soybean divergence. The expression levels of two common bean FDH genes under different treatments were investigated by quantitative RT-PCR analysis. FDH genes are differentially up-regulated after biotic and abiotic stresses (infection with the fungus Colletotrichum lindemuthianum, and dark treatment, respectively). The present study provides the first report of FDH transcript accumulation after biotic stress, suggesting the involvement of FDH in the pathogen resistance process.

Molecular cloning and expression analysis of the pathogenesis-related gene VaPR2 in azuki bean (Vigna angularis)

A cDNA sequence including the complete open reading frame was isolated from leaves of flowering azuki bean variety No. 56. The nucleotide sequence was submitted to the NCBI Genbank database. The gene was related to pathogenesis (protein gene resistance to disease) and named VaPR2. VaPR2 was very similar to PvPR2, PvPR1, and CpPR3, with identity ranging from 88.4% to 91.1%. The gene was highly induced after challenging with the soybean mosaic virus and the anthracnose fungus Colletotrichum lindemuthianum . Results from RT-PCR analysis showed that the VaPR2 gene is expressed strongly in stem, leaves, flowers, and pods, but weakly in roots.