Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare

Colletotrichum gloeosporioides f. sp. malvae, isolate Biomal®, ATCC 20767, was originally isolated from round-leaved mallow (Malva pusilla) and developed as a weed biocontrol agent. Ribosomal DNA sequence analysis was recently used to re-classify this fungus as C. orbiculare, which is an aggregate species with a number of formae speciales. Several morphological features of ATCC 20767 were examined that were consistent with those described for C. orbiculare, and inoculation of a number of Nicotiana species and several cultivars of N. tabacum showed that this fungus was pathogenic to many of these previously undescribed hosts. Spore germination and appressorium formation were higher on tobacco than previously observed on round-leaved mallow. The pathogen produced melanized appressoria on N. tabacum leaves that formed preferentially at the anticlinal epidermal cell wall. A symptomless phase of infection persisted for 72–96 h postinoculation, during which time the fungus first produced a spherical infection vesicle from an infection peg, and then large primary hyphae which grew through the epidermal cells. The large primary hyphae were highly constricted at the points of penetration of the host cell walls. Thin secondary hyphae appeared at 96–120 h postinoculation coinciding with the appearance of light green, water-soaked spots and the formation of acervuli. The infection of tobacco by C. orbiculare ATCC 20767 is not a non-specific interaction but appears to follow an intracellular hemibiotrophic infection process that is very similar to that established for the C. orbiculare infection of round-leaved mallow, cucurbits and beans.     

Formation of highly branched hyphae by Colletotrichum acutatum within the fruit cuticles of Capsicum spp.

This study showed that Colletotrichum acutatum penetrates the cuticle layer of Capsicum spp. fruits by forming a previously uncharacterized structure from appressoria. This unusual structure was localized in the cuticle layer. The structure, formed within 24 h post‐inoculation (hpi), was a highly branched, well‐differentiated hypha which penetrated the epidermal cell at 72 hpi. The novel structure, with abnormally thick walls (about 250 nm), often formed multiple branches in the affected chilli pepper. This dendroid structure, probably required for penetration, was formed exclusively in the cuticle layer of chilli pepper fruits and was not found when C. acutatum was inoculated onto pepper petals, mango leaves, or fruits of tomato and aubergine. Colletotrichum acutatum produced similar dendroid structures within resistant chilli pepper fruits, but eventually these structures turned dark brown and no further infection in the epidermal cells occurred, implicating the presence of inhibitors of the formation and development of the dendroid penetration structure in the resistant line.     

The spore coat of the bean anthracnose fungus Colletotrichum lindemuthianum is required for adhesion, appressorium development and pathogenicity

The spores (conidia) of the bean anthracnose fungal pathogen, Colletotrichum lindemuthianum, adhere to the aerial parts of plants to initiate the infection process. In previous studies we have shown that the Colletotrichum spores are surrounded by a fibrillar spore coat, comprising several major glycoproteins. Previous evidence showed that a monoclonal antibody (UB20) that recognised these glycoproteins was able to inhibit adhesion of spores to a hydrophobic surface. In this paper we have further studied the role of the spore coat in adhesion, germination and fungal development by studying the effects of UB20 and protease treatment of spores. The latter treatment has previously been shown to remove the spore coat. Spores germinate on glass, polystyrene and water agar, however, appressoria only develop on glass or polystyrene, showing a requirement for a hard surface. Removal of the spore coat with protease inhibits adhesion at 30 min, before the secretion of ECM glycoproteins. Protease treatment also inhibits the development of appressoria and reduces pathogenicity on leaves. Incubation of spores with the MAb UB20 inhibits adhesion at 30 min, but does not affect appressorium formation or pathogenicity. The results suggest that an intact spore coat has two functions; it is required for adhesion to a hydrophobic surface and for the detection of a hard surface necessary for appressorium formation. We suggest that contact with a hard surface, rather than adhesion, is the key event leading to appressorium formation.     

Impact of tricyclazole and azoxystrobin on growth,sporulation and secondary infection of the rice blast fungus,Magnaporthe oryzae

BACKGROUND: Rice blast, caused by Magnaporthe oryzae B. Couch sp. nov., is one of the most destructive rice diseases worldwide, causing substantial yield losses every year. In Italy, its management is based mainly on the use of two fungicides, azoxystrobin and tricyclazole, that restrain the disease progress. The aim of this study was to investigate and compare the inhibitory effects of the two fungicides on the growth, sporulation and secondary infection of M. oryzae. RESULTS: Magnaporthe oryzae mycelium growth was inhibited at low concentrations of azoxystrobin and relatively high concentrations of tricyclazole, while sporulation was more sensitive to both fungicides and was affected at similarly low doses. Furthermore, infection efficiency of conidia obtained from mycelia exposed to tricyclazole was affected to a higher extent than for conidia produced on azoxystrobin‐amended media, even though germination of such conidia was reduced after azoxystrobin treatment. CONCLUSIONS: This study presents for the first time detailed azoxystrobin and tricyclazole growth–response curves for M. oryzae mycelium growth and sporulation. Furthermore, high efficacy of tricyclazole towards inhibition of sporulation and secondary infection indicates an additional possible mode of action of this fungicide that is different from inhibition of melanin biosynthesis. Copyright © 2012 Society of Chemical Industry     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.     

Isolation,production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases

Antibiosis has been shown to be an important mode of action by Trichoderma species used in the protection of grapevine pruning wounds from infection by trunk pathogens. The major active compound from Trichoderma isolates known to protect grapevine pruning wounds from trunk pathogen infection was isolated and identified. The compound, a 6‐pentyl‐α‐pyrone (6PP), was found to be the major secondary metabolite, by quantity, which accumulated in the culture filtrate of T. harzianum isolate T77 and the two T. atroviride isolates UST1 and UST2. Benzimidazole resistant mutants generated from these isolates also produced 6PP as their main secondary metabolite, except for a mutant of T77 that had lost its ability to produce 6PP. The isolates UST1 and UST2 were co‐cultured with the grapevine trunk pathogens Eutypa lata and Neofusicoccum parvum in a minimal defined medium and a grapevine cane‐based medium (GCBM). Co‐culturing UST1 with N. parvum induced 6PP production in the minimal defined medium and the GCBM. The production of 6PP by UST2 was induced in the GCBM, while co‐culturing with the two trunk pathogens either reduced or had no effect on 6PP production. Mycelial growth and ascospore/conidia germination of E. lata, N. australe, N. parvum and Phaeomoniella chlamydospora were inhibited by 6PP in a concentration‐dependent manner. The results show that the presence of N. parvum and grapevine wood elicits the production of 6PP, suggesting that this metabolite is involved in Trichoderma–pathogen interactions on grapevine pruning wounds.