Ana B. Menéndez

Hyphal interaction between Trichoderma harzianum and Sclerotinia sclerotiorum and its role in biological control

Abstract Hyphal interactions between the mycoparasite Trichoderma harzianum (BAFC Cult. No. 72) and the soilborne plant pathogenic fungus Sclerotinia sclerotiorum were investigated in dual culture and in sterilized soil, by light and scanning electron microscopy. In dual culture, T. harzianum hyphae grew towards and coiled around the S. sclerotiorum hyphae. Dense coils of hyphae of T. harzianum and partial degradation of the Sclerotinia cell wall were observed in later stages of the parasitism. In sterile soil, conidia of T. harzianum germinated and the developing mycelium made contact with that of S. sclerotiorum, forming short branches and appressorium-like bodies which aided in holding and penetrating the host cell wall. An in vitro system was developed to test the ability of T. harzianum to control Sclerotinia wilt in cucumber and lettuce: coating seeds with T. harzianum conidia reduced the pre- and post-emergence effect of S. sclerotiorum in cucumber by 69 and 80%, respectively, and in lettuce by 46 and 72%, respectively. In the greenhouse, the disease caused by S. sclerotiorum in lettuce was reduced by treating seedlings with a peat-bran preparation of T. harzianum. Despite the non-significance of the reduction in disease, Trichoderma-treated lettuce seedlings were much more developed than controls. In sunflower, significant reductions (in the range of 68 to 84%) in disease incidence were obtained by incorporating the peat-bran T. harzianum preparation into the seedling rooting mixture. Hyphal mycoparasitism, rather than sclerotial parasitism, is suggested to be the mechanism by which T. harzianum controls S. sclerotiorum under these conditions.

Mycelial compatibility groups in Buenos Aires field populations of Sclerotinia sclerotiorum (Sclerotiniaceae)

One-hundred and forty isolates of Sclerotinia sclerotiorum (Lib.) DeBary were obtained in a 6500-km2 area of the Buenos Aires Province. These isolates were collected from soybean (60 isolates), lettuce (59 isolates) and sunflower (21 isolates) fields. Fifty different mycelial compatibility groups (MCGs) were distinguished overall, 27 of which consisted of two or more isolates. Populations of S. sclerotiorum were made up by numerous MCGs and shared a similar frequency profile regardless the locality or the date of sampling. MCG occurrences differed among the crops, only two MCGs were shared by the three crops and approximately 60% of the MCGs were unique for each crop. The Shannon diversity index (Ho) of MCG for the whole region was 0.314 (Htot). Partition of total diversity (Htot) showed that 98.4% corresponded to variations of diversity within populations. Morphological characteristics were not significantly different among MCGs, except for the dry weight per sclerotium. On the basis of the detached celery petiole assay, there were no differences in aggressiveness between MCGs either. However, isolates collected from sunflower plants were more aggressive than those collected from soybean, regardless the MCG they belonged. Aggressiveness was positively correlated to colony radial growth. After principal component analyses (PCA) were performed, it could be identified a main central group of isolates and two other smaller groups. Main contributors to the total variance were the percentages of large sclerotia, the dry weight per sclerotium and the percentages of medium and small sclerotia.

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742). Biological control of Sclerotinia sclerotiorum by Trichoderma harzianum.

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source.

Photosynthetic responses mediate the adaptation of two Lotus japonicus ecotypes to low temperature

Lotus species are important forage legumes due to their high nutritional value and adaptability to marginal conditions. However, the dry matter production and regrowth rate of cultivable Lotus spp. is drastically reduced during colder seasons. In this work, we evaluated the chilling response of Lotus japonicus ecotypes MG-1 and MG-20. No significant increases were observed in reactive oxygen species and nitric oxide production or in lipid peroxidation, although a chilling-induced redox imbalance was suggested through NADPH/NADP(+) ratio alterations. Antioxidant enzyme catalase, ascorbate peroxidase, and superoxide dismutase activities were also measured. Superoxide dismutase, in particular the chloroplastic isoform, showed different activity for different ecotypes and treatments. Stress-induced photoinhibition also differentially influenced both ecotypes, with MG-1 more affected than MG-20. Data showed that the D2 PSII subunit was more affected than D1 after 1 d of low temperature exposure, although its protein levels recovered over the course of the experiment. Interestingly, D2 recovery was accompanied by improvements in photosynthetic parameters (Asat and Fv/Fm) and the NADPH/NADP(+) ratio. Our results suggest that the D2 protein is involved in the acclimation response of L. japonicus to low temperature. This may provide a deeper insight into the chilling tolerance mechanisms of the Lotus genus.