Visualization of Three Sclerotiniaceae Species Pathogenic on Onion Reveals Distinct Biology and Infection Strategies

Botrytis squamosa, Botrytis aclada, and Sclerotium cepivorum are three fungal species of the family Sclerotiniaceae that are pathogenic on onion. Despite their close relatedness, these fungi cause very distinct diseases, respectively called leaf blight, neck rot, and white rot, which pose serious threats to onion cultivation. The infection biology of neck rot and white rot in particular is poorly understood. In this study, we used GFP-expressing transformants of all three fungi to visualize the early phases of infection. B. squamosa entered onion leaves by growing either through stomata or into anticlinal walls of onion epidermal cells. B. aclada, known to cause post-harvest rot and spoilage of onion bulbs, did not penetrate the leaf surface but instead formed superficial colonies which produced new conidia. S. cepivorum entered onion roots via infection cushions and appressorium-like structures. In the non-host tomato, S. cepivorum also produced appressorium-like structures and infection cushions, but upon prolonged contact with the non-host the infection structures died. With this study, we have gained understanding in the infection biology and strategy of each of these onion pathogens. Moreover, by comparing the infection mechanisms we were able to increase insight into how these closely related fungi can cause such different diseases.

The Botrytis cinerea Crh transglycosylae is a cytoplasmic effector triggering plant cell death and defense response

Crh proteins catalyze crosslinking of chitin and glucan polymers in the fugal cell wall. We revealed a novel and unexpected role of Botrytis cinerea BcCrh1 as a cytoplasmic effector and elicitor of plant defense. During saprophytic growth the BcCrh1 protein is localized in vacuoles and ER. Upon plant infection the protein accumulates to high levels in infection cushions, it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids were found sufficient for protein uptake and cell death induction, respectively. Dimerization of BcCrh proteins was necessary for the transglycosylation activity and proper fungal development, while the monomeric proteins was sufficient for induction of cell death. Arabidopsis lines expressing the bccrh1 gene had reduced sensitivity to B. cinerea, demonstrating the potential use of the protein in plant immunization against necrotrophic pathogens.

Influence of Different Nitrogen Sources on Growth and Pathogenic Capability of Rhizoctonia solani Causing Root Rot of Faba Bean

The effect of different nitrogen sources (glucosamine sulfate, ammonium sulfate, aspartic acid, phenylalanine and peptone) in comparison to sodium nitrate, the major nitrogen compound in basal agar Czapek’s synthetic medium growth were studied on the linear growth of Rhizoctonia solani and its pathogenicity on faba bean germinated seeds. Ammonium sulfate exhibited faster liner growth and showed the same effect as the basal medium with sodium nitrate while glucosamine sulfate showed less growth rate compared with sodium nitrate. Glucosamine sulfate and ammonium sulfate showed a significant reduction in number of infection cushions which led to significant decrease of disease index in vitro. Under greenhouse conditions, glucosamine sulfate or peptone as a sole nitrogen sources in food requirements of Rhizoctonia solani inoculum depressed the virulence of the fungus. The effect of different amounts of glucosamine sulfate was determined on fungal growth rate, infection cushions, disease index in vitro and polyphenol oxidase activity. Increasing amount of glucosamine sulfate showed significant reduction of growth rate in comparison to the basal medium with sodium nitrate. All seeds subjected to R. solani grown on different amount of glucosamine sulfate showed the lower number of infection cushions, disease index and polyphenol oxidase activity compared with sodium nitrate. Under greenhouse conditions, disease index showed a significantly decreased effect when glucosamine sulfate used as soil applications and showed better effect on shoot weight and root weight compared with control plants treated with sodium nitrate. Our study proposes that glucosamine sulfate may act as controlling factor of pathogenicity genes of R. solani

Responses of rice to Rhizoctonia solani and its toxic metabolite in relation to expression of Osmyb4 transcription factor

The reaction of IR 50, TRY 3, and IR 36 cultivars of rice to R. solani challenge, the causal agent of sheath blight, and its toxic metabolite was studied. Differential response of these cultivars to the pathogen and/or toxin inoculation was observed in detached leaf sheaths and greenhouse-grown plants. The observations were based on disease scoring, electrolyte leakage, and also microscopic views of infection cushions. The Osmyb4 gene expression was studied in the tissues from all these experiments and a correlation between the level of expression and disease response of the varieties was found at least in some experiments. The mechanisms regulated by Osmyb4 might have a lower but significant contribution to the tolerance of rice cultivars to sheath blight.

First Report of Violet Root Rot Caused by Rhizoctonia crocorum on Alfalfa in Iran

In August 2000, violet root rot of alfalfa (Medicago sativa L.) was observed in circular to irregular patches of alfalfa. Affected alfalfa plants turned yellow to brown in fields in the Zanjan Province of Iran. Diseased roots had a compact felt cloak of violet mycelium with white or purple strands. Samples of symptomatic roots were collected from 11 locations in the province and cultured on potato dextrose agar and malt-extract agar. The morphology of fungi isolated from pieces of alfalfa root with infection cushions from each location was consistent with Rhizoctonia crocorum (Pers.:Fr.) DC. (teleomorph Helicobasidium brebissonii (Desm.) Donk) (1). Seeds of alfalfa cv. Hamadani were sown in soil infested with inoculum of one isolate of R. crocorum prepared on a sandrye grain-water medium (20 g per pot) (2). Under 10 h of light at 22°C, destroyed taproots were evident after 8 months on 64 of 100 inoculated plants, and R. crocorum was reisolated from all 64 plants. Fifty noninoculated plants showed no symptoms. This is the first report of violet root rot of alfalfa in Iran. References: (1) P. Roberts. Pages 157–158 in: Rhizoctonia-Forming Fungi. The Trustees, Royal Botanic Garden, Kew, UK, 1999. (2) P. G. Valder. Tran. Br. Mycol. Soc. 41:283, 1958.

Association of Binucleate Rhizoctonia with Soybean and Mechanism of Biocontrol of Rhizoctonia solani

The association of binucleate Rhizoctonia (BNR) AG-K with soybean and the interaction of BNR, R. solani AG-4, and soybean seedlings were investigated to elucidate the mechanism of biocontrol of R. solani by BNR. Sixty-hour-old seedlings were inoculated and incubated in a growth chamber at 24°C; plants were examined with light microscopy and with scanning and transmission electron microscopy at various times following inoculation. BNR grew over hypocotyls, roots, and root hairs, but only colonized epidermal cells. Hyphae of BNR appeared to attach to the epidermis and, 5.5 h following inoculation, began penetrating cells by means of penetration pegs without forming distinct appressoria or infection cushions. There was evidence of cuticle degradation at the point of penetration. Infection hyphae moved to adjacent epidermal cells by direct penetration of epidermal radial walls. There were epidermal and cortical cell necrosis, beginning with the fragmentation of the tonoplast and followed by the disintegration of cytoplasm, organelles, and plasma membranes. Cell necrosis was also observed in adjacent cells where there was no evidence of BNR hyphae. Cell walls were not destroyed. After 144 h, there was noevidence of BNR hyphae in cortical cells. Attempted penetrations were observed, but papillae formed on the inside of cortical cell walls. Pre-inoculation of soybean seedlings with BNR 24 or 48 h before inoculation with R. solani (1 cm between inocula) affected the growth of R. solani on soybean tissue. There were fewer hyphae of R. solani, the hyphae branched sparingly, and infection cushions were rare when compared with hyphal growth on soybean inoculated only with R. solani. These effects were observed before the BNR hyphae began to intermingle with the hyphae of R. solani on the surface of the inoculated host. Preinoculation of soybean seedlings 24 h before inoculation with R. solani significantly (P = 0.05) reduced disease incidence and severity caused by R. solani AG-4. The lesions caused by R. solani always appeared distally, not proximally, to the BNR inoculum. The interactions of intermingling hyphae of BNR and R. solani were examined in vitro and on the surface of the host. There was no evidence of lysis, mycoparasitism, inhibition of growth, or any other form of antagonism between hyphae. The results of these studies strongly suggest that induced resistance is the mechanism of biocontrol of R. solani on soybean by BNR. The inhibition of hyphal growth of R. solani on the surface of soybean tissue preinoculated with BNR appears to be a novel characteristic of induced resistance.