Study on Trichoderma Citrinoviride 31/4 – Antagonistic Activity and Jasmonic Acid

The genus Trichoderma was first identified by the German scientist Persoon in 1974, and there are currently 89 species in the genus. Trichoderma fungi often break down organic compounds in the soil into minerals and plays an important role in soil fertility formation. Trichoderma citrinoviride 31/4 isolated from the soil forms a colony with light yellow-green mycelium on the surface of the PDA medium, with spherical spore, septate hyphae, branched conidophores. Trichoderma citrinoviride 31/4 had antagonistic activity of 66.7% against Cladosporium fulvum, 77.5% against Alternaria alternata, and 59.1% against Fusarium oxysporium after 7 days. Experimental results showed that the progress of tomato alternariosis treated with Trichoderma citrinoviride 31/4 was neutralized from the 7th day, and the biological activity was averagely 74% on the 21st day. Trichoderma citrinoviride 31/4 contains 0.0135 mg / kg of jasmonic acid in plants treated with 109 cell / ml and 0.0076 mg/kg in plants treated with 108 cell / ml.

Screening new Trichoderma isolates for antagonistic activity against several phytopathogenic fungi, including Fusarium spp.

Aims. To obtain and characterize new isolates of Trichoderma antagonistic to phytopathogenic fungi, including Fusarium spp., and 2) to determine their suitability for mass production under different cultivation conditions. Methods. Microbiological, cultural-morphological, statistical. Results. From plants affected by phytopathogenic fungi: cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), white cabbage (Brassica oleracea L.), winter wheat (Triticum aestivum L.), spring barley (Hordeum vulgare L.) in the Forest-Steppe of Ukraine (Kyiv re- gion) 11 new Trichoderma isolates were obtained. Preliminary, morphological determination allocated fi ve of them to T. viride (isolates CK, 165, 27, 49, 35), two of them to T. koningii (21, 64) and four of them to T. longibrachiatum (161, 162, 163, 164). All isolates showed moderate to high antagonistic activity towards 8 phytopathogenic fungal species (Fusarium oxysporum, Fusarium solani, Alternaria cucumerina, Colletotrichum phomoides, Botrytis cinerea, Trichothecium roseum, Penicillium sp., Cladosporium fulvum). In a dual culture experiment they showed generally similar or often higher activity to the above-mentioned fungi than the 8 control strains used in our study, belonging to T. viride (5 strains), T. koningii (2 strains) and T. harzianum (1 strain), which have been maintained since long time in our laboratory. The most active new isolate CK, (presumably) T. viride, showed comparable high activity towards all phytopathogenic fungi as compared to our most active control strain of T. viride, no. 23. The latter is the basis of a biocide Trichodermin, produced by biolaboratories of Ukraine, including the Institute of Plant Protection, NAAS, Kyiv. Chlamydospore production of all isolates and strains studied in submerged culture varied from 10 6 to 3 · 10 7 spores/ml, were T. viride isolates and strains were on the higher end. Isolates of ‘T. longibrachiatum’ did not produce chlamydospores in submerged culture. Upon superfi cial cultivation on barley grain, the strains and isolates of T. viride were also characterized by the highest production of spores (6 · 10 9 -9 · 10 9 spores/g) as compared to those of T. kon- ingii, T. harzianum (5.5 · 10 9 -6.8 · 10 9 spores/g) and T. longibrachiatum (1.3 · 10 8 -6.8 · 10 8 spores/g). In an in-vivo experiment under laboratory conditions the most promising antagonistic isolate CK was used to inoculate wheat seed and tested for protection against Fusarium root rot (inoculum a mixture of F. avenaceum, F. culmorum, F. gibbosum, F. oxysporum, in 4·10 4 spores/g), where it gave an 83 % reduction in root rot as compared to the non-inoculated con- trol. Conclusions. Five new isolates preliminarily (on the basis of morphological characteristics only) allocated to T. viride and four to T. longibrachiatum demonstrated in vitro the highest and widest antagonistic activity against the phytopathogenic fungal species Fusarium oxysporum, Fusarium solani, Alternaria cucumerina, Colletotrichum pho- moides, Botrytis cinerea, Trichothecium roseum, Penicillium sp., Cladosporium fulvum, as compared to new isolates, preliminarily allocated to - T. harzianum and T. koningii. New isolate CK (allocated to T. viride) showed a promising and similar high antagonistic activity as compared to our T. viride 23 strain, which is already successfully used in the biocide Trichodermin. Since this isolate CK also produced a high number of chlamydospores in submerged culture (3 · 10 7 spores/ml) and conidia (8 · 10 9 spores/g) when surface cultured on barley grain respectively, it is a potential new candidate for a biocide. When this CK isolate was studied in a small laboratory pot experiment, to control Fu- sarium root rot in wheat by preventive seed inoculation, it caused an 83 % reduction in this Fusarium root rot. Its usefulness under fi eld conditions and its effect on growth of plants will be investigated in future research

Extracellular proteolytic cascade in tomato activates immune protease Rcr3

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogenCladosporium fulvum (syn.Passalora fulva). Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 ofP. infestanscan block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation inNicotiana benthamianarequires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.

Genes Encoding Recognition of the Cladosporium fulvum Effector Protein Ecp5 Are Encoded at Several Loci in the Tomato Genome

The molecular interactions between tomato and Cladosporium fulvum have been an important model for molecular plant pathology. Complex genetic loci on tomato chromosomes 1 and 6 harbor genes for resistance to Cladosporium fulvum, encoding receptor like-proteins that perceive distinct Cladosporium fulvum effectors and trigger plant defenses. Here, we report classical mapping strategies for loci in tomato accessions that respond to Cladosporium fulvum effector Ecp5, which is very sequence-monomorphic. We screened 139 wild tomato accessions for an Ecp5-induced hypersensitive response, and in five accessions, the Ecp5-induced hypersensitive response segregated as a monogenic trait, mapping to distinct loci in the tomato genome. We identified at least three loci on chromosomes 1, 7 and 12 that harbor distinct Cf-Ecp5 genes in four different accessions. Our mapping showed that the Cf-Ecp5 in Solanum pimpinellifolium G1.1161 is located at the Milky Way locus. The Cf-Ecp5 in Solanum pimpinellifolium LA0722 was mapped to the bottom arm of chromosome 7, while the Cf-Ecp5 genes in Solanum lycopersicum Ontario 7522 and Solanum pimpinellifolium LA2852 were mapped to the same locus on the top arm of chromosome 12. Bi-parental crosses between accessions carrying distinct Cf-Ecp5 genes revealed putative genetically unlinked suppressors of the Ecp5-induced hypersensitive response. Our mapping also showed that Cf-11 is located on chromosome 11, close to the Cf-3 locus. The Ecp5-induced hypersensitive response is widely distributed within tomato species and is variable in strength. This novel example of convergent evolution could be used for choosing different functional Cf-Ecp5 genes according to individual plant breeding needs.