Appressorial Formation Research Articles

Development of a Robust Screening Method for Pathogenicity of Colletotrichum spp. on Strawberry Seedlings Enabling Forward Genetic Studies.

Generation and screening for nonpathogenic mutants is a popular tool for identifying pathogenicity-related genes. Successful application of this technique for plant fungal pathosystems requires reliable and rapid screening procedures. This study reports on the development of a rapid in vitro bioassay enabling large-scale screening and isolation of nonpathogenic mutants of Colletotrichum gloeosporioides and C. acutatum on strawberry seedlings. Inoculation was carried out on strawberry seedlings at two different developmental stages: 12-week-old (young) and 15-week-old (older) seedlings. A comparison was made between two inoculation techniques, (i) foliar dip and (ii) root soak, at two incubation temperatures (19 and 25°C). Mortality of young seedlings was observed 4 days after inoculation with both species, reaching 50% within 10 days, using both techniques at 25°C. However, mortality of older seedlings was delayed by 4 days compared with that in the young seedlings when using the root-soak method. Disease development decreased in young and older seedlings at the lower temperature. This method also was reliable in determining pathogenicity of the cucurbit-specific C. magna that did not cause disease symptoms on strawberry by either inoculation method. The proposed method enabled screening of more than 980 restriction enzyme-mediated integration mutants resulting in a selection of five reduced-virulence isolates. Initial characterization of some of these mutants revealed large differences in germination and appressorial formation compared with pathogenic isolates.

Detection of fungi producing infection-inhibiting metabolites against Alternaria alternata Japanese pear pathotype from fungi inhabiting internal tissues of Japanese pear shoots

Fungi inhabiting Japanese pear were isolated from internal tissues of cv. Nijisseiki, and culture filtrates (CFs) of 100 isolates were evaluated for their inhibitory activity against infection by Alternaria alternata Japanese pear pathotype. CFs of 11 isolates inhibited lesion formation on the pear by the pathogen. Among these isolates, CFs of five isolates inhibited spore germination. CFs of the six other isolates inhibited appressorial formation, infection hypha formation, AK-toxin production, or a combination of these actions. Analysis of sequence homology in the rDNA ITS1 regions of these isolates showed that most isolates had high homology with some fungal endophytes.

Pyricularia setariae: a potential bioherbicide agent for control of green foxtail (Setaria viridis)

One hundred and thirty-three fungal isolates, pathogenic to green foxtail, were evaluated for weed control potential under controlled conditions. To determine weed control efficacy, these pathogens were applied as spore or mycelial suspensions at approximately 105propagules ml−1to green foxtail at the three-leaf stage. One week after inoculation, most isolates caused only minor injury to the plants, but 15 isolates caused 50 to 100% disease. Among the most efficacious isolates, only those ofPyricularia setariaeexhibited strong host specificity to the target weed, revealing no significant pathogenicity on 28 other plant species tested, including many important crops such as wheat, barley, and oat. On green foxtail leaves, conidia of this fungus germinated readily at 14, 20, and 26 C, but the process of germination and appressorial formation was more rapid at the higher temperatures. The fungus applied at the concentration of 105spores ml−1reduced weed fresh weight by 34% 7 d after the treatment when compared with controls, whereas a concentration of 107spores ml−1reduced fresh weight by 87%. This efficacy was comparable with that of the herbicide sethoxydim. When applied to the weed at the one- to four-leaf stages, the fungus reduced green foxtail fresh weight by more than 80%. Efficacy was slightly lower on plants at the five-leaf stage or older. On the green foxtail biotype resistant to the herbicide sethoxydim,P. setariaecaused 80% fresh weight reduction compared with untreated controls, as opposed to 17% achieved with the herbicide. At 20 C, the fungus required a minimum of 6-h dew period to initiate infection, but a 10-h dew period was needed to cause severe damage to green foxtail.

Specific inhibition of spore germination of Alternaria brassicicola by fistupyrone from Streptomyces sp. TP-A0569

Fistupyrone (FP), a metabolite from Streptomyces sp. TP-A0569, inhibited the in vivo infection of Chinese cabbage seedlings by Alternaria brassicicola. To detect the possible action sites of FP, the effect of FP on the infection behavior of A. brassicicola and A. alternata was investigated. When spores of A. brassicicola were suspended in FP solution and inoculated on host leaves, FP at 0.1 ppm significantly inhibited spore germination, appressorial formation, and infection hypha formation of A. brassicicola. Host-specific AB-toxin production and lesion formation by A. brassicicola spores were also reduced significantly by treatment with FP 1 ppm. The effect of FP seemed to be irreversible because significant washing of FP-treated spores with distilled water (DW) did not change the inhibitory effects. In contrast, A. alternata isolates such as Japanese pear pathotype, apple pathotype, and saprophyte behaved almost equally in both FP- and DW-treated spores. Mycelial dry weight in potato dextrose broth and mycelial diameters on potato dextrose agar, gelatin glucose agar, and Czapek solution agar of both A. brassicicola and A. alternata were not different with or without addition of FP. These results indicate that FP at low concentrations has a fungicidal effect on spores of A. brassicicola but not on spores of A. alternata; FP also does not affect the vegetative phase of these fungi.

Infection behavior of Alternaria alternata Japanese pear pathotype and localization of 1,3-β- d -glucan in compatible and incompatible interactions between the pathogen and host plants

The infection behavior of Alternaria alternata Japanese pear pathotype and host responses were examined cytologically and ultrastructurally in compatible and incompatible interactions between Japanese pear plants and the pathogen during the infection process. No differences in spore germination, appressorial formation, or cuticle penetration were detected between susceptible and resistant leaves inoculated with spores of the pathogen. On the other hand, infection hyphae emerged from penetration pegs and invaded the pectin layers of susceptible leaves but not those of resistant leaves. The results indicate that expression of resistance to the pathogen may be induced before the formation of infection hyphae in pear tissues. Plasma membrane modifications were observed ultrastructurally 3 h after inoculation only in susceptible leaf cells. Papillae were usually formed in epidermal cells underneath the appressoria in resistant leaves at the stage when the progress of the pegs was stopped 9 h after inoculation. Callose deposition was observed fluorescence-microscopically as papillae at the infection sites in abaxial epidermis of both cultivars. The immunoelectron microscopic results demonstrated that the components of the papillae and the extracellular polysaccharides that had accumulated in plasma membrane modification sites were compatible with 1,3-β-d-glucan. These results suggest that the glucan deposition results from host cuticle damage by fungal invasion and from plasma membrane damage by AK-toxin.

Evidence that the appressorial development in barley powdery mildew is controlled by MAP kinase activity in conjunction with the cAMP pathway

Development of the barley powdery mildew fungus involves the sequential formation of a primary germ tube, an appressorial germ tube, and an appressorium. Previously, we have shown that the cAMP pathway controls the emergence of the two germ tubes. Following identification of two MAP kinase genes in an EST database from developing conidia we studied the role of the MAP kinase pathway and its interaction with the cAMP pathway. Fungal MAP kinase activity increased rapidly during mildew development, reaching a maximum between 2 and 8 h after inoculation. Sphingosine or PAF-16, activators of the MAP kinase pathway, increased activity and appressorial development whilst an inhibitor, PD 98059, decreased both. Studies on the interaction between the cAMP and MAPK pathways revealed that several effectors of the MAPK pathway had no effect on cAMP levels. However upstream effectors of the cAMP pathway, such as cholera toxin and pertussis toxin (activators of G α proteins) increased MAPK activities whereas downstream effectors such as forskolin (adenylyl cyclase activator) or H89 (PKA inhibitor) had no effect. Combined application of forskolin and sphingosine produced a rise in appressorial germ tube and appressorial formation higher than when either pathway was stimulated individually. These results suggest that the two pathways cooperate in appressorial development.

The effect of low temperatures on Colletotrichum acutatum and Colletotrichum gloeosporioides causing body rots of avocados in New Zealand

The effect of extended periods at low temperatures on spore germination, survival and appressorial formation in Colletotrichum acutatum and C. gloeosporioides, and on pre-formed appressoria of C. gloeosporioides, was studied on water agar on glass slides on a temperature gradient plate. C. acutatum was more tolerant of low temperatures than was C. gloeosporioides. The addition of peptone to simulate endogenous nutrients on the avocado fruit surface affected spore germination and survival only slightly, and the effect was not statistically significant. These trends in vitro were also apparent in isolations from fruit rots. Infections by C. acutatum were not reduced by storage at 5.5°C before ripening. Incidence of infections caused by C. gloeosporioides was significantly fewer in fruit coolstored at 5.5°C for 30 days prior to ripening at 20°C than in fruit stored at 20°C. In the two lines of fruit studied, coolstorage reduced body rots in one line but not the other. The rots on the line of fruit not affected by coolstorage were mainly caused by C. acutatum. These results suggest that in coolstored fruit, the reduction in incidence of rots caused by C. gloeosporioides, but not of rots caused by the closely related fungus C. acutatum, was due to the greater sensitivity of spores of C. gloeosporioides to cold temperatures. Survival of pre-formed appressoria of C. gloeosporioides was not affected by temperatures used for coolstorage of avocados.

Mechanisms involved in control of Blumeria graminis f.sp. hordei in barley treated with mycelial extracts from cultured fungi

Treatment with mycelial extracts, prepared from liquid cultures of Bipolaris oryzae, Pythium ultimum and Rhizopus stolonifer, protected barley (Hordeum vulgare) against powdery mildew disease caused by the fungus Blumeria graminis f.sp. hordei. The mechanisms of this protection were studied using histopathological methods and molecular analysis. Germination and appressorial formation of B. graminis were generally reduced after treatment with mycelial extracts. Although this reduction (between 12 and 62% depending on treatment and experiment) was inconsistent and only occasionally significantly different from the water‐treated control, it indicated a direct antifungal effect of the extracts. In situations where the fungus succeeded in forming an appressorium, penetration efficiency and haustorium formation from these appressoria was not affected – no enhanced penetration resistance associated with papilla formation was detected. However, a post‐penetration effect was observed, as B. graminis colonies on mycelial extract‐treated leaves produced 50% fewer hyphae than on controls. Northern blot analyses showed earlier accumulation of defence‐related gene transcripts following treatment with B. oryzae and P. ultimum mycelial extracts, and to a lesser extent R. stolonifer mycelial extract, compared with water‐treated leaves. It is suggested that the protection mechanism of the mycelial extracts involves direct antifungal effects and possible induced resistance for the B. oryzae and P. ultimum mycelial extracts.

Factors Affecting Sporulation, Germination, and Appressoria Formation of Epicoccosorus nematosporus as a Mycoherbicide Under Controlled Environments

To develop Epicoccosorus nematosporus as a mycoherbicide of Eleocharis kuroguwai, the optimum temperature and humidity for sporulation of the pathogen were studied. Conidial production was most abundant at <TEX>$28^{\circ}C$</TEX> with RH 60%, which yielded 661 mg in 9 cm Petri dish. Light intensity of 3,000 up to 7,500 lux was effective in stimulating conidial production of E. nematosporus on oatmeal agar, Light intensity affected sporulation more significantly than temperature. In the pot test, at least 12 h of dew period at <TEX>$20^{\circ}C$</TEX> and <TEX>$25^{\circ}C$</TEX> was required to achieve satisfactory conidial germination and appressorial formation. Few were killed at 8 h of dew period regardless of temperature. Sixteen hours of a single dew treatment immediately after inoculation killed more plants than did two or three repetitive dew treatments of 8-12 h.

Formation of appressoria by two species of lepidopteran-pathogenic Entomophthorales

Fungal pathogens frequently form appressoria, specialized hyphal swellings on the surfaces of hosts. Production of appressoria by two entomophthoralean species that infect lepidopteran larvae, Entomophaga maimaiga Humber, Shimazu &amp; Soper and Furia gastropachae (Raciborski) Filotas, Hajek &amp; Humber, was investigated in vitro 6–8 h after conidial discharge. Entomophaga maimaiga appressoria were elongate, irregularly swollen structures located adjacent to the conidium or at the ends of short germ tubes. The highest percentages of appressoria were formed on hard surfaces such as polystyrene (21.5 ± 4.6%) and mylar (22.2 ± 4.3%). Nutrients or chemical stimuli were not required for appressorial formation but could stimulate growth as germ tubes. The contribution of surface hydrophobicity to appressorium formation was questionable; while appressoria were formed on hydrophobic surfaces, they were also formed to a lesser extent on glass, which is hydrophilic. When conidia of F. gastropachae were exposed to similar substrates and conditions, appressoria were never made, supporting the hypothesis that stimuli for appressorium formation can be species specific.Key words: appressorium, entomopathogenic fungi, infection process, Entomophthorales.

Evidence for a role of cutinase in pathogenicity of Pyrenopeziza brassicae on brassicas

Supernatants from apple cutin-induced cultures of the light leaf spot pathogen Pyrenopeziza brassicae were shown to contain a single esterase with a pI of approx. 4.4 and a MW of 21kDa. Analysis of the hydrolysis products derived from enzymatic cleavage of3H labelled cutin by TLC demonstrated that these supernatants had cutinolytic activity. Treatment with the serine esterase inhibitors ebelactones A and B resulted in 50% inhibition of theP. brassicae esterase activity at concentrations of 4 and 0.4μ gml−1, respectively. It is therefore concluded that following induction in vitro with apple cutin, P. brassicae produces a single extracellular esterase with cutinolytic activity.A putative cutinase gene was cloned by heterologous Southern hybridization of P. brassicae genomic DNA using the Botrytis cinerea cutinase gene as a probe. The ORF (706bp) is predicted to encode a polypeptide of 203 amino acids with a molecular weight of 20.379kDa and a pI of 4.38. This polypeptide shows high levels of homology with the cutinases of B. cinerea and of other fungi, and contains the conserved histidine, aspartic acid and serine catalytic triad. Expression of the putative cutinase gene was shown to correlate with induction of esterase activity in vitro.SEM analysis of Brassica napus leaves infected withP. brassicae confirmed that direct penetration of the host surface occurs with no evidence of appressorial formation. The ebelactones A (100μ gml−1) and B (10μ gml−1) had no effect on germination of P. brassicae conidia in vitro; however, a marked decrease in pathogenicity of P. brassicae was observed on B. napus plants treated with 100μ gml−1ebelactone A or 10μ gml−1ebelactone B. In these plants, failed penetration was observed and the number of acervular conidiomata was greatly reduced. These results suggest a functional role for fungal cutinolytic activity in pathogenicity of P. brassicae on B. napus.

Evaluation of Infection Processes and Resulting Disease Caused byDendryphion penicillatumandPleospora papaveraceaonPapaver somniferum

ABSTRACT Two pathogenic fungi of opium poppy, Pleospora papaveracea and Dendryphion penicillatum, were isolated from field material in Beltsville, MD. The processes of infection by these two fungi were studied to determine the optimal environmental conditions for infection. Both fungi formed appressoria capable of penetrating directly through the plant epidermal layer. Of the two fungi, P. papaveracea was more aggressive, causing more rapid necrosis. Appressorial formation by P. papaveracea occurred as early as 4 h after application of a conidial suspension to poppy leaves. P. papaveracea formed more appressoria than did D. penicillatum, especially at cool temperatures (7 to 13 degrees C). In greenhouse studies, P. papaveracea caused more damage to opium poppy than did D. penicillatum when applied in 10% unrefined corn oil. In the field, P. papaveracea was more consistent in its effects on opium poppy from a local seed source designated Indian Grocery. P. papaveracea caused higher disease ratings, more stem lesions, and equal or greater yield losses than did D. penicillatum on Indian Grocery. The late-maturing opium poppy variety White Cloud was severely damaged by disease, regardless of formulation or fungal treatment. P. papaveracea was the predominant fungus isolated from poppy seed capsules and the only fungus reisolated from the field the following year. These studies provide a better understanding of the infection process and the differences between these two pathogenic fungi and will be beneficial for the development of the fungi as biological control agents.

(+)-Catechin Acts as an Infection-Inhibiting Factor in Strawberry Leaf

ABSTRACT An infection-inhibiting factor (IIF) was isolated from strawberry leaves and identified as (+)-catechin. This compound inhibited the formation of infection hyphae from appressoria of Alternaria alternata, but allowed both spore germination and appressorial formation. It is a normal component of strawberry leaves, but further accumulates as the major IIF in response to inoculation with nonpathogenic spores of A. alternata. The accumulation of (+)-catechin on a susceptible host was not induced, however, by inoculation with pathogenic spores of the strawberry pathotype or by inoculation with nonpathogenic spores supplemented with host-specific toxin (AF-toxin I). These results imply that (+)-catechin acts as a protective agent during induced resistance and that AF-toxin I acts as a fungal suppressor of induced resistance.

Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew.

Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner.

Survival of Oidium anacardii on cashew (Anacardium occidentale) in southern Tanzania

During March and April of 1993 and 1994, surveys on the incidence and severity of cashew powdery mildew (Oidium anacardii) were conducted in the Newala, Mtwara, Nachingwea and Tunduru areas of southern Tanzania to determine the variation in perennation between localities. Only immature cashew shoots, panicles and fruit can be infected by O. anacardii conidia. Cashew trees at sites in each district were assessed for shoot and panicle production and cashew powdery mildew. Survival of O. anacardii between seasons, in any area, was determined by the degree of production of shoots that were within the canopy and by the incidence of infection. Immature shoots produced from the main branches within the tree canopy were the main source of active powdery mildew in all districts; trees in the Newala district had the highest numbers of infected immature shoots in comparison with survey sites in the other areas. During the 1994 cashew‐growing season (June–August), powdery mildew developed more rapidly and affected more shoots on the inside of the tree canopy than on the outside. Germination of conidia was reduced after aqueous suspension for 3 h. Germination on cashew leaves submerged under 2 mm of water was not affected. Appressorial and hyphal formation by germinating conidia on leaves decreased with increasing duration under water. Germination of conidia on glass slides at 100% r.h. was higher at 25 and 30°C than at 15°C and there was no germination at 35°C.

Differential Interactions of aColletotrichum gloeosporioides isolate with green and red pepper fruits

Differential interactions ofColletotrichum gloeosporioides isolate KG 13 with green and red pepper fruits (Capsicum annuum were found when it was inoculated on unwounded and wounded fruits. The isolate produced the typically necrotic, sunken anthracnose symptom on unwounded and wounded green fruits, and wounded red ones, but not on unwounded red ones. Appressorial formation of the fungus on the surfaces of compatible green fruits was higher than on incompatible red ones up to 12 h after inoculation. More and longer infection pegs from appressoria were produced on green than on red fruits. When cuticular wax layers of green and red fruits were removed by dipping in chloroform, red ones only produced larger lesions and more conidia than water-dipped controls did. However, differences in lesion diameter and conidial production were not observed between green and red fruits wounded by pin-pricking. In addition, concentrations of wax extracted from the surface of green and red fruits affected conidial germination and appressorial formation of the fungus. These findings suggest that the isolate KG 13 ofC. gloeosporioides may react differentially to green and red pepper fruits, probably due to the physical and chemical differences in cuticular layers of the fruits.

Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic.

Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthracnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, including germination and subsequent differentiation, resulting in appressorium formation. Our previous studies with pharmacological effectors of signaling pathways have suggested the involvement of cyclic AMP (cAMP)-dependent protein kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) subunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene replacement. Southern blot analysis with C. trifolii genomic DNA suggested that Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RNA from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactivated by gene replacement, the transformants showed a small reduction in growth relative to the wild type and conidiation patterns were altered. Importantly, PKA-deficient strains were unable to infect intact alfalfa (host) plants, though only a slight delay was observed in the timing for conidial germination and appressorial formation in the Ct-PKAC disruption mutants. Moreover, these mutants were able to colonize host tissues following artificial wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely due to a failure in penetration. Our results demonstrate that PKA has an important role in regulating the transition between vegetative growth and conidiation, and is essential for pathogenic development in C. trifolii.

Molecular cloning and characterization of Ct-PKAR, a gene encoding the regulatory subunit of cAMP-dependent protein kinase in Colletotrichum trifolii.

Colletotrichum trifolii is a plant pathogenic fungus causing alfalfa anthracnose. Prepenetration development, including conidial germination and appressorial formation, are requisite for successful infection. Pharmacological data from our laboratory indicated a role for a cAMP-dependent protein kinase (PKA) pathway during these early morphogenic transitions. Thus, the cloning and characterization of the genes for PKA catalytic and regulatory subunits were undertaken to more precisely determine the function of PKA during C. trifolii pathogenic growth and development. In this report, the cloning, sequencing, and partial characterization of the gene encoding the regulatory subunit of cAMP-dependent protein kinase (Ct-PKAR) is described. An open reading frame of 1,212 bp containing 404 predicted amino acid residues was identified. Database analysis revealed that the deduced amino acid sequence of Ct-PKAR shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. Furthermore, the Ct-PKAR protein is classified as a type II regulatory subunit based on the presence of the hall-mark autophosphorylation site. Southern blot analysis indicated that Ct-PKAR is a single-copy gene. Northern blot analysis showed that the expression of Ct-PKAR is developmentally regulated. Ct-PKAR was shown to be a functional regulatory subunit of PKA by complementating the Neurospora crassa mcb mutant, which has a temperature-sensitive mutation in the regulatory subunit of PKA.

Target Site of a New Antifungal Compound KC10017 in the Melanin Biosynthesis ofMagnaporthe grisea

A new experimental fungicide KC10017 (3-[4′-bromo-2′, 6′-dimethylphenoxy]methyl-4-[(3″-methylphenyl) aminocarbonyl]methyl-1,2,4-oxadiazol-5-one) completely controlled rice blast disease at more than 1.0 μg/ml without curative activity. When the chemical was applied after the inoculation was made with injury, the fungicide had no effect on controlling rice blast. The fungicide at concentrations of more than 1 μg/ml blocked appressorial melanization and penetration of cellophane membrane byMagnaporthe griseaP-2 without affecting fungal growth, spore germination, and appressorial formation. To elucidate the target site of KC10017 in melanin biosynthesis inhibition,M. griseaP-2 was grown in potato dextrose broths treated with both KC10017 and tricyclazole. The mycelia and culture media of cultures treated with both KC10017 and tricyclazole at a concentration of 5 μg/ml each became colorless like as the cultures treated with KC10017 alone, whereas the tricyclazole-treated cultures exhibited red color. Shunt products of 3,4-dihydro-3,4,8-trihydroxy-1-(2H)-naphthalenone, 3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone, 3,4-dihydro-4,6,8-trihydroxy-1-(2H)-naphthalenone, and 4-hydroxyscytalone were absent or present in only trace amounts in the cultures treated with KC10017 alone or both KC10017 and tricyclazole. However, they were detected at much higher levels in the cultures treated with tricyclazole alone. On the other hand, appressorial melanization and penetration ability inM. griseatreated with KC10017 was recovered by the addition of scytalone. These results demonstrate that the target site of KC10017 in melanin biosynthesis inhibition is at the pentaketide synthesis step and/or at the pentaketide cyclization step prior to 1,3,6,8-tetrahydroxynaphthalene formation.

The role ofPseudomonas spp. and competition for carbon, nitrogen and iron in the enhancement of appressorium formation byColletotrichum coccodes on velvetleaf

Colletotrichum coccodes is currently being investigated as a mycoherbicide against the weed velvetleaf (Abutilon theophrasti). Two isolates ofPseudomonas spp. (Ps2 and Ps5) reduced the percentage of germ tubes and increased appressorial formation ofC. coccodes on detached leaves of velvetleaf. A study was conducted to see whether this effect could be attributed to competition for nutrients or iron betweenC. coccodes andPseudomonas spp. Ps2 and Ps5 had no effect on early spore germination, but reduced the percentage of germ tubes at 24 and 30 h, compared to the nontreated control. This reduction was diminished by the addition of nutrients but not Fe3+. Ps2 and Ps5 stimulated the formation of dark-coloured appressoria without germ tubes (AWGT), but this stimulation was diminished by the addition of nutrients or Fe3+. Germ tube branching at 30 h was also inhibited by the bacteria, but was not diminished by the addition of nutrients or iron. EDTA stimulated conidial germination at 10 h, which was reduced by the addition of Fe3+. However, EDTA did not stimulate the formation of appressoria (AWGT). These results suggest that the reduction in the percentage of germ tubes and the increase in the percentage of appressoria induced by the bacteria may be due to the competition for carbon or nitrogen. Iron competition may also be involved in the stimulation of appressorial formation, but not in the reduction in germ tube percentage and branching. Phylloplane bacteria may compete for carbon, nitrogen and iron, limiting the saprophytic phase of the pathogen on the phylloplane and accelerating the development of the parasitic phase. This may enhance the field efficacy ofC. coccodes as a biocontrol agent against velvetleaf.