Cryptic peptides (cryptides) are small bioactive molecules generated via degradation of functionally active proteins. Only a few examples of plant cryptides playing an important role in plant defense have been reported to date, hence our knowledge about cryptic signals hidden in protein structure remains very limited. Moreover, little is known about how stress conditions influence the size of endogenous peptide pools, and which of these peptides themselves have biological functions is currently unclear. Results: Here, we used mass spectrometry to comprehensively analyze the endogenous peptide pools generated from functionally active proteins inside the cell and in the secretome from the model plant Physcomitrella patens. Overall, we identified approximately 4,000 intracellular and approximately 500 secreted peptides. We found that the secretome and cellular peptidomes did not show significant overlap and that respective protein precursors have very different protein degradation patterns. We showed that treatment with the plant stress hormone methyljasmonate induced specific proteolysis of new functional proteins and the release of bioactive peptides having an antimicrobial activity and capable to elicit the expression of plant defense genes. Finally, we showed that the inhibition of protease activity during methyl jasmonate treatment decreased the secretome antimicrobial potential, suggesting an important role of peptides released from proteins in immune response. Conclusions: Using mass-spectrometry, in vitro experiments and bioinformatics analysis, we found that methyl jasmonate acid induces significant changes in the peptide pools and that some of the resulting peptides possess antimicrobial and regulatory activities. Moreover, our study provides a list of peptides for further study of potential plant cryptides.

Colletotrichum coccodes is a plant pathogenic fungus affecting different organs of potato, tomato, and some other plants. The leaf infection with C. coccodes may result in a rapid development of infection nidus and high losses caused by the infection of tomato fruits and potato tubers during harvesting. Our study showed the presence of C. coccodes on tomato and potato leaves with dry necrotic lesions. The presence of a DNA region specific for C. coccodes was detected in DNA samples isolated from infected tomato leaves collected in the Rostov region (7 and 1 samples from two distinct fields) and the Krasnodar Territory (5 samples from one field). In the case of potato leaf samples, the frequency of C. coccodes detection was lower than in tomato leaves. This fungus was revealed only in five potato leaf samples: one from the Northern Ossetia, one from the Kostroma region, and three from the Mariy El Republic. Pure cultures of two isolates were isolated from a naturally infected potato leaf. According to morphological criteria, size of spores and sclerotia, habitus of colonies, and species-specific DNA sequences, both strains completely corresponded to C. coccodes.

Alternaria species are the causal agents of potato and tomato early blight disease. Three species-specific PCR primer sets were designed for Alternaria alternata sensu lato, Alternaria solani and Alternaria infectoria identification. The specificity of primers was confirmed by the absence of amplified products after PCR with DNA of Alternaria spp., other potato and tomato pathogenic fungal species and plant DNA. Constructed primers were applied for the survey of early blight agents on affected potato and tomato leaves collected in different regions of Russia. All three Alternaria pathogens were present in blighted leaves alone or in complex (A. alternata + A. solani, A. infectoria + A. alternata and A. solani + A. infectoria). Alternaria alternata (sensu lato) was the most abundant species. It was detected in 50% of tested potato leaves and 41% of tested tomato leaves (alone or in complex). Alternaria solani was found in 30 and 35% leaves, respectively. Alternaria infectoria occurred in potato and tomato samples occurred at a somewhat lower frequency (13 and 8%, respectively). Using the specific primers, it was possible to exclude the Alternaria disease in some leaves with symptoms similar to early blight.

Helminthosporium solani strains were isolated from potato tubers that were collected in Russia or taken from the imported German and Dutch seed tubers. Sequencing of the nuclear ribosomal genes and internal transcribed spacers (ITS) was done for all 16 tested strains. Obtained sequences for all strains were identical and had 100% similarity to the sequences from GenBank identified as Helminthosporium solani. The obtained molecular data confirmed the morphological identification based on the width and length of conidia, the shape of conidiophores and the colony morphology. Screening for resistance to the fungicides Score 250 SC (active ingredient difenoconazole 250 g/l), Quadris (azoxistrobin 250 g/l), Tecto 500 SC (thiabendazole 500g/l), Zeroxxe (colloidal silver particles (3 g/l) stabilized with amphoteric surfactant) was done. Pure cultures of the fungal strains were placed in the centre of Petri dishes containing malt agar with a fungicide at the rates of 0.1; 1; 10; 100 and 1000 mg/l (accounted for the concentration of the active ingredient). Malt agar without the fungicide was used as the control. Growth inhibition of 50% (EC50) compared to the control was detected based on the dose-response curves. Difenoconazole (EC50<0.12 mg/l) and colloidal silver (EC50<76 mg/l) were the most effective fungicides. No strains resistant to the aforementioned fungicides were found. In most cases, azoxistrobin was effective against H. solani (EC50< 7mg/l), but there were several strains with resistance to this fungicide (EC50>100mg/l). Thiabendazole appears to be effective against the sensitive strains of H. solani (EC50 < 7,3 mg/l); however, 5 studied strains from Russia and the Netherlands were found to be extremely resistant to it (EC50>1000mg/l). The sequence of their β-tubulin gene contains a SNP mutation in 198 codon or 200 codon, translating to Gln (CAG) instead of Glu (GAG) or Tyr (TAC) instead of Phe (TTC), respectively. Thus, the resistance of the Russian, European, and American strains has the identical genetical background and is conferred by the same mutations.

Sustainable agriculture calls for minimal use of agrochemicals in order to protect the environment. It has caused an increase in the rate of nanoparticles use, in particular silver nanoparticles (AgNPs) due to their safety for mammals, unique biological activity and a broad spectrum of action against fungal and bacterial pathogens. Until now the use of AgNPs dispersions in the agricultural sector has been essentially limited due to many factors decreased their stability (mixing with other pesticides, presence of electrolytes).We present a versatile synthesis of polyampholyte surfactant (tallow amphopolycarboxyglycinate)stabilized AgNPs.We took a close look at unique aggregation behavior (via dynamic light scattering andUV–vis spectroscopy) and biocidal activity of obtained silver colloids. AgNPs are characterized by exclusively high aggregative stability in the presence of coagulating agentsNaNO3 and NaSO4 (up to 1M), during drying/redispergation, and frost/defrost cycles. The dispersion of AgNPs shows high biocidal activity (EC50 is ten times lower than commercial species ones) with respect to Phytophthora infestans and phytopathogenic fungi. This points to the possibility of successful application of silver preparations within agriculture with the goal of partial reduction of the use of toxic and expensive synthetic antibiotics and pesticides.

Fungicide difenoconazole is applied for field treatment to protect potato and tomato against early blight, caused by the Alternaria fungi. It is practically important to estimate the impact of difenoconazole application against late blight pathogen Phytophthora infestans (Mont.) de Bary because of early and late blight often come together on potato. Effect of fungicide difenoconazole on the radial growth of colony of P. infestans and the formation of oospores in agar media and in detached leaves was investigated. There was no significant influence of difenoconazole on the radial growth of P. infestans colony, small inhibition occurred only at high concentration of fungicide (100 mg/l). Difenoconazole inhibited the oospore formation in the medium with positive dependence on concentrations of fungicide and temperature increasing. Tested in leaves difenoconazol inhibited the oosporogenesis with positive dependence on concentrations of fungicide also. The tiers of the plant were considered as source of oospores with different potential. In laboratory conditions (test on detached leaves) the higher production of oospores was detected in leaves from middle and low tiers rather than from top tiers.

Populations of the potato and tomato late blight pathogen from different regions of the European part of Russia were studied for their genotypic structure and metalaxyl resistance. The majority of the studied populations showed a high genotypic diversity, i.e. strains of A1 and A2 mating types, Ia and IIa mitochondrial DNA haplotypes, and different genotypes at Pep1 and Pep2 loci were identified. The highest genotypic diversity was registered among P. infestans populations from the North Caucasus, Mariy El Republic, and the Moscow region. The obtained data confirm a high probability of crossing and hybridization within the most part of the field populations of P. infestans from the European part of Russia. Most of the studied populations included strains with high and low metalaxyl resistance, the incidence of resistant strains depending on the use of metalaxyl-containing fungicides.

Phytophthora infestans samples were collected during 1997and 1998 at multiple sites in Russia from Sakhalin Island in the Far East across Siberia (nine sites, 160 isolates) to the Moscow region (four sites, 325 isolates). Additionally, 12 isolates that were obtained previously were analyzed. All isolates were analyzed for mating type, and sensitivity to metalaxyl. Isolates from within any of the nine sites outside of the Moscow region were monomorphic for mating type and nearly monomorphic for metalaxyl resistance. In contrast, both A1 and A2 isolates were detected in the Moscow region, and these isolates were also polymorphic for metalaxyl resistance. In two sites in Siberia only A2 mating type strains were detected, in the other six sites in Siberia and in Sakhalin Island, only A1 mating types were detected. A subset of isolates (n=191) was also analyzed for pathotype. All isolates were highly complex (many compatibilities, with a mean value of compatibility of ca 8.4 (max = 10). All isolates (n = 43) from Sakhalin Island were compatible with all 10 of the R-genes tested. A further subset of isolates (n= 70, including 12 isolates collected before 1997) was analyzed for genotype at the Glucose-6-phosphate isomerase and Peptidase loci, mtDNA haplotypes, and RFLP pattern using the RG57 probe. The US-1 clonal lineage (previously dominant) was not detected in the 1997-1998 sample. The population of P. infestans near Moscow in 1997 and 1998 was highly diverse with 15 unique genotypes (including both mating types) among a sample of 18 isolates. In contrast the populations of P. infestans in Siberia had limited diversity, with only three multilocus genotypes detected and most populations were dominated by the SIB-1 clonal lineage. This lineage accounted for 31of the 39 strains collected in Siberia that were assayed for multilocus genotype.