The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum

Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, Clpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.     

Nonpathogenic Strains of Colletotrichum lindemuthianum Trigger Progressive Bean Defense Responses during Appressorium-Mediated Penetration

The fungal bean pathogen Colletotrichum lindemuthianum differentiates appressoria in order to penetrate bean tissues. We showed that appressorium development in C. lindemuthianum can be divided into three stages, and we obtained three nonpathogenic strains, including one strain blocked at each developmental stage. H18 was blocked at the appressorium differentiation stage; i.e., no genuine appressoria were formed. H191 was blocked at the appressorium maturation stage; i.e., appressoria exhibited a pigmentation defect and developed only partial internal turgor pressure. H290 was impaired in appressorium function; i.e., appressoria failed to penetrate into bean tissues. Furthermore, these strains could be further discriminated according to the bean defense responses that they induced. Surprisingly, appressorium maturation, but not appressorium function, was sufficient to induce most plant defense responses tested (superoxide ion production and strong induction of pathogenesis-related proteins). However, appressorium function (i.e., entry into the first host cell) was necessary for avirulence-mediated recognition of the fungus.     

Dose responses for Colletotrichum lindemuthianum elicitor-mediated enzyme induction in French bean cell suspension cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 g elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 g ml^-1. The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; -methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.Abbreviations PAL phenylalanine ammonia-lyase - PMS Phytophthora megasperma var sojae     

Production of a cell wall-associated endopolygalacturonase by Colletotrichum lindemuthianum and pectin degradation during bean infection

The bean pathogen Colletotrichum lindemuthianum expresses two endopolygalacturonase genes, CLPG1 and CLPG2, during interaction with its host plant. However, only CLPG1 was found to be secreted to the extracellular medium during saprophytic growth of the fungus on pectin. To localize CLPG2, a FLAG epitope sequence was inserted in the C-terminal sequence of CLPG2 and the modified gene was introduced into C. lindemuthianum. Western blot analysis using a FLAG monoclonal antibody allowed the detection of CLPG2 in intracellular protein extracts and in the cell wall fraction, but not in the culture medium. Indirect immunofluorescence microscopy was performed to detect CLPG2 during saprophytic or parasitic growth. According to the expression pattern of CLPG2, it was found that CLPG2 accumulates in the fungal cell wall during growth on pectin medium and during appressorium formation, both in vitro and during interaction with the plant. Pectin degradation was not detected around the infection peg using the monoclonal antibody JIM7, specific for methyl-esterified galacturonan. However, extensive pectin dissolution was observed during the development of secondary hyphae.     

Quantitative analysis of race-specific resistance to Colletotrichum lindemuthianum in common bean

Molecular genetic maps continue to play a major role in breeding of crop species. The common bean genetic map of the recombinant inbred line population IAC-UNA × CAL 143 (UC) has been used to detect loci controlling important agronomic traits in common bean. In the current study, new microsatellite markers were added to the UC map and the linkage analysis was refined using current genomic resources of common bean, in order to identify quantitative resistance loci (QRL) associated with different races of the anthracnose pathogen. A single race inoculation was conducted in greenhouse using four plants per plot. Both race-specific and joint-adjusted disease severity means, obtained from linear-mixed model, were used to perform multiple interval mapping (MIM) and multi-trait MIM (MTMIM). In total, 13 and 11 QRL were identified by MIM and MTMIM analyses, respectively; with nine being observed in both analyses. ANT02.1^UC and ANT07.1^UC showed major effects on resistance both for MIM and MTMIM. Common major QRL for resistance to the three anthracnose races were expected, since high genetic pairwise-correlation was observed between the race-specific and joint-adjusted disease severity means. Therewith, both ANT02.1 and ANT07.1 can be regarded as valuable targets for marker-assisted selection; and so, putative genes potentially involved in the resistance response were identified in these QRL regions. Minor effect QRL were also observed, showing differential affects either on race-specific or multi-trait analyses and may play a role on durable horizontal resistance. These results contribute to a better understanding of the host-pathogen interaction and to breeding for enhancing resistance to Colletotrichum lindemuthianum in common bean.     

Biochemical changes in French bean pods infected with Colletotrichum lindemuthianum*

Infection of bean pods with Colletotrichum lindemuthianum leads to the appearance in diffusates of a range of fluorescent and phenolic compounds and of at least two inhibitory compounds. These compounds were found to be absent or in reduced concentration in control diffusates, although the inhibitors were frequently observed to appear at low concentrations without infection. Although sugars and amino acids were found to be released into diffusates, variation in the concentration of these stimulants was found to have little effect on spore germination. Evidence from solvent partition, spectrophotometry and chromatography suggests that the two inhibitors can be tentatively identified with inhibitors previously described from French bean, and both appear to be phenolic substances. It is suggested that inhibitor production may be regarded as part of a general change in aromatic biosynthesis following infection.     

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.     

Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection

The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.     

Inhibition of photosynthesis by Colletotrichum lindemuthianum in bean leaves determined by chlorophyll fluorescence imaging

Infection of bean leaves by Colletotrichum lundemuthianum causes vein necrosis and subsequent localized wilting of the blade. The effect of infection on photosynthesis was investigated by imaging leaf chlorophyll fluorescence as a means of mapping stomatal and metabolic inhibition of photosynthesis. During infection, CO₂ assimilation (An), stomatal conductance to water vapour, and photosynthetic electron transport rate (J_t) decreased, whereas dark respiration increased. An decreased more than was expected from the reduction in green leaf area, showing that photosynthesis was inhibited in apparently healthy areas. Under subsaturating irradiance, images of J_t in air showed that photosynthesis decreased gradually, with this effect shifting from green to necrotic areas. Sudden increase in CO₂ concentration to 0·74% in the atmosphere around the leaf only partially reversed this inhibition, showing that both stomatal and metabolic inhibition occurred. Under limiting irradiance, decreases in J_t and in maximal J_t during high CO₂ exposure as leaf damage severity increased suggested that metabolic inhibition was mediated through an inhibition of Ribulose 1·5‐bisphosphate (RuBP) regeneration. Finally, the importance of our data in terms of assessing the loss of photosynthetic yield from visible symptoms – as is currently performed in epidemiology – is discussed.     

Immunomagnetic purification of Colletotrichum lindemuthianum appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

A specific inhibitor of Colletotrichum lindemuthianum protease from kidney bean (Phaseolus vulgaris) seeds

A specific protein—an inhibitor of Colletotrichum lindemuthianum protease—was isolated from kidney bean seeds in a homogeneous form. The purification procedure included gel filtration, isoelectric focusing and affinity chromatography on trypsin-Sepharose column. The latter was introduced to separate the fungal protease inhibitor from closely related trypsin and chymotrypsin inhibitors present in kidney bean seeds.     

Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

Application of endophytic bacteria for controlling anthracnose disease (Colletotrichum lindemuthianum) on bean plants

Over 100 endophytic bacterial isolates were isolated from surface-sterilised roots of the Fabaceae family in East Azerbaijan farms. These isolates were screened for their in vitro biocontrol activity against Colletotrichum lindemuthianum by dual culture technique using potato dextrose agar (PDA) medium. Eight bacterial isolates (Bacillus subtilis subsp. subtilis, Bacillus atrophaeus, B. tequilensis, B. subtilis subsp. spizizenii, Streptomyces cyaneofuscatus, S. flavofuscus, S. parvus, S. acrimycini) showed promising inhibition on mycelial growth of C. lindemuthianum , and thus, these isolates were selected for greenhouse experiments. The disease control rate using these selected endophytic bacteria was varied from 40 to 76.80% in greenhouse without any negative effects on different growth performance, suggesting that these selected endophytic bacteria are potential to be developed as biocontrol agents.     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

Antibiosis of Streptomyces griseus Against Colletotrichum lindemuthianum

Streptomyces griseus produces an antibiotic substance against Colletotrichum lindemuthianum because, in dual culture on potato dextrose agar (PDA), a large inhibitory zone was formed between the colonies of S. griseus and C.lindemuthianum. When S. griseus was placed adjacent to a colony of C. lindemuthianum in PDA, S.griseus grew poorly in PDA but it thrived and sporulated profusely when it came in contact with a colony of C. lindemuthianum indicating that S.griseus might require some essential nutrients from C.lindemuthianum. Scanning electron microscopy showed that hyphae of S.griseus in contact with C.lindemuthianum produced appressorium-like swellings or simply grew on the hyphal surface of C.lindemuthianum. Internal parasitism was evidenced by the presence of hyphae and conidia of the mycoparasite inside the host hyphae. Internal parasitism resulted in formation of several blebs. Cell walls of the parasitized hyphae degenerated having a sponge-like texture. The blebs broke off and left rounded holes on the hyphae.     

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.     

Molecular Detection of Colletotrichum lindemuthianum by Duplex PCR

A duplex PCR technique was developed to detect the pathogenic fungus Colletotrichum lindemuthianum infection in the tissues of common bean. Based on the differences of 24 internal transcribed spacer, DNA sequences of Colletotrichum spp. retrieved from GeneBank database, one pair of specific primers of CY1/CY2 (CY1: 5'-CTT TGT GAA CAT ACC TAA CC-3'; CY2: 5'-GGT TTT ACG GCA GGA GTG-3'), was designed. The CY1/CY2 primers amplified a single PCR product of 442 bp only from C. lindemuthianum and Colletotrichum orbiculare , not from any other tested species. By using random amplification of polymorphic DNA technique, a product closely associated with C. lindemuthianum was generated. This product was cloned, sequenced and used for designing a species-specific primers of CD1/CD2 (CD1: 5'-ACC TGG ACA CAT AAG TCA AAG-3'; CD2: 5'-CAA CAA TGC CAG TAT CAG AG-3'). The CD1/CD2 primers could distinguish C. lindemuthianum from C. orbiculare by a 638 bp PCR band. A duplex PCR method, combining both primers of CY1/CY2 and CD1/CD2, was used to detect C. lindemuthianum infection. The sensitivity of the detection with this PCR method was 1 pg of pure genomic DNA from the pathogen. Therefore, the PCR-based methods could be used for accurate and rapid detection of C. lindemuthianum from common bean.     

B1-phytoprostanes trigger plant defense and detoxification responses

Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B(1)-phytoprostanes (PPB(1)) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB(1)-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB(1) regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB(1) considerably prevented cell death caused by severe CuSO(4) poisoning. Several Arabidopsis genes induced by PPB(1), such as those coding for adenylylsulfate reductase, tryptophan synthase beta-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB(1) treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB(1). In three different plant species, PPB(1)-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB(1)-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB(1) represent stress signals that improve plants capacity to cope better with a variety of stresses.