Pythium oligandrum, strain M1, is a soil oomycete successfully used as a biological control agent (BCA), protecting plants against fungal, yeast, and oomycete pathogens through mycoparasitism and elicitor-dependent plant priming. The not yet described Pythium strains, X42 and 00X48, have shown potential as BCAs given the high activity of their secreted proteases, endoglycosidases, and tryptamine. Here, Solanum lycopersicum L. cv. Micro-Tom seeds were coated with Pythium strains, and seedlings were exposed to fungal pathogens, either Alternaria brassicicola or Verticillium albo-atrum. The effects of both infection and seed-coating on plant metabolism were assessed by determining the activity and isoforms of antioxidant enzymes and endoglycosidases and the content of tryptamine, amino acids, and heat shock proteins. Dual culture competition testing and microscopy analysis confirmed mycoparasitism in all three Pythium strains. In turn, seed treatment significantly increased the total free amino acid content, changing their abundance in both non-infected and infected plants. In response to pathogens, plant Hsp70 and Hsp90 isoform levels also varied among Pythium strains, most likely as a strategy for priming the plant against infection. Overall, our results show in vitro mycoparasitism between Pythium strains and fungal pathogens and in planta involvement of heat shock proteins in priming.

Biopreparáty spol s r o Tylišovská 1 160 00 Prague 6 Czech Republic

Department of Analytical Chemistry Faculty of Science Charles University Hlavova 2030 128 43 Prague 2 Czech Republic

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 128 43 Prague 2 Czech Republic

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Ons L., Bylemans D., Thevissen K., Cammue B.P.A. Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control. Microorganisms. 2020;8:1930. doi: 10.3390/microorganisms8121930. PubMed DOI PMC

Mandelc S., Javornik B. The secretome of vascular wilt pathogen Verticillium albo-atrum in simulated xylem fluid. Proteomics. 2015;15:787–797. doi: 10.1002/pmic.201400181. PubMed DOI

Barrit T., Porcher A., Cukier C., Satour P., Guillemette T., Limami A.M., Teulat B., Campion C., Planchet E. Nitrogen nutrition modifies the susceptibility of Arabidopsis thaliana to the necrotrophic fungus, Alternaria brassicicola. Physiol. Plant. 2021;174:e13621. doi: 10.1111/ppl.13621. PubMed DOI

Tralamazza S.M., Piacentini K.C., Iwase C.H.T., Rocha L.D. Toxigenic Alternaria species: Impact in cereals worldwide. Curr. Opin. Food Sci. 2018;23:57–63. doi: 10.1016/j.cofs.2018.05.002. DOI

Belonoznikova K., Hyskova V., Chmelik J., Kavan D., Cerovska N., Ryslava H. Pythium oligandrum in plant protection and growth promotion: Secretion of hydrolytic enzymes, elicitors and tryptamine as auxin precursor. Microbiol. Res. 2022;258:126976. doi: 10.1016/j.micres.2022.126976. PubMed DOI

Benhamou N., le Floch G., Vallance J., Gerbore J., Grizard D., Rey P. Pythium oligandrum: An example of opportunistic success. Microbiology. 2012;158:2679–2694. doi: 10.1099/mic.0.061457-0. PubMed DOI

Gerbore J., Benhamou N., Vallance J., Le Floch G., Grizard D., Regnault-Roger C., Rey P. Biological control of plant pathogens: Advantages and limitations seen through the case study of Pythium oligandrum. Environ. Sci. Pollut. Res. Int. 2014;21:4847–4860. doi: 10.1007/s11356-013-1807-6. PubMed DOI

McGowan J., Fitzpatrick D.A. Genomic, network, and phylogenetic analysis of the oomycete effector arsenal. mSphere. 2017;2:e00408-17. doi: 10.1128/mSphere.00408-17. PubMed DOI PMC

Derevnina L., Dagdas Y.F., De la Concepcion J.C., Bialas A., Kellner R., Petre B., Domazakis E., Du J., Wu C.H., Lin X., et al. Nine things to know about elicitins. New Phytol. 2016;212:888–895. doi: 10.1111/nph.14137. PubMed DOI

Takenaka S., Nishio Z., Nakamura Y. Induction of defense reactions in sugar beet and wheat by treatment with cell wall protein fractions from the mycoparasite Pythium oligandrum. Phytopathology. 2003;93:1228–1232. doi: 10.1094/PHYTO.2003.93.10.1228. PubMed DOI

Takenaka S., Tamagake H. Foliar spray of a cell wall protein fraction from the biocontrol agent Pythium oligandrum induces defence-related genes and increases resistance against Cercospora leaf spot in sugar beet. J. Gen. Plant Pathol. 2009;75:340–348. doi: 10.1007/s10327-009-0186-9. DOI

Takenaka S., Yamaguchi K., Masunaka A., Hase S., Inoue T., Takahashi H. Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato. J. Plant Physiol. 2011;168:1972–1979. doi: 10.1016/j.jplph.2011.05.011. PubMed DOI

Ai G., Yang K., Tian Y., Ye W., Tian Y., Du Y., Zhu H., Li T., Xia Q., Shen D., et al. Prediction and characterization of RXLR effectors in Pythium species. Mol. Plant Microbe Interact. 2019;33:1046–1058. doi: 10.1094/MPMI-01-20-0010-R. PubMed DOI

Yang K., Dong X., Li J., Wang Y., Cheng Y., Zhai Y., Li X., Wei L., Jing M., Dou D. Type 2 Nep1-like proteins from the biocontrol oomycete Pythium oligandrum suppress Phytophthora capsici infection in Solanaceous plants. J. Fungi. 2021;7:496. doi: 10.3390/jof7070496. PubMed DOI PMC

Hase S., Shimizu A., Nakaho K., Takenaka S., Takahashi H. Induction of transient ethylene and reduction in severity of tomato bacterial wilt by Pythium oligandrum. Plant Pathol. 2006;55:537–543. doi: 10.1111/j.1365-3059.2006.01396.x. DOI

Hase S., Takahashi S., Takenaka S., Nakaho K., Arie T., Seo S., Ohashi Y., Takahashi H. Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato. Plant Pathol. 2008;57:870–876. doi: 10.1111/j.1365-3059.2008.01858.x. DOI

Lou B.-G., Wang A.-Y., Lin C., Xu T., Zheng X.-D. Enhancement of defense responses by oligandrin against Botrytis cinerea in tomatoes. Afr. J. Biotechnol. 2011;10:11442–11449. doi: 10.5897/AJB11.618. DOI

Takenaka S. Studies on biological control mechanisms of Pythium oligandrum. J. Gen. Plant Pathol. 2015;81:466–469. doi: 10.1007/s10327-015-0620-0. DOI

Le Floch G., Rey P., Benizri E., Benhamou N., Tirilly Y. Impact of auxin-compounds produced by the antagonistic fungus Pythium oligandrum or the minor pathogen Pythium group F on plant growth. Plant Soil. 2003;257:459–470. doi: 10.1023/A:1027330024834. DOI

Doubnerova V., Ryslava H. Roles of Hsp70 in plant abiotic stress. In: Gaur R.K., Sharma P., editors. Molecular Approaches in Plant Abiotic Stress. CRC Press; Boca Raton, FL, USA: 2014. pp. 44–66.

Song Z., Pan F., Lou X., Wang D., Yang C., Zhang B., Zhang H. Genome-wide identification and characterization of Hsp70 gene family in Nicotiana tabacum. Mol. Biol. Rep. 2019;46:1941–1954. doi: 10.1007/s11033-019-04644-7. PubMed DOI

Sappah A.E., Abbas M., Elrys A.S., Yadav V., El-Sappah H.H., Zhu Y., Huang Q., Yu W., Soaud S.A.E., Xianming Z., et al. The Hsp70 gene family in Solanum lycopersicum; Genome-wide identification and expression analysis under heavy metals stresses. Res. Sq. 2021 doi: 10.21203/rs.21203.rs-546628/v546621. in press . DOI

Zai W.S., Miao L.X., Xiong Z.L., Zhang H.L., Ma Y.R., Li Y.L., Chen Y.B., Ye S.G. Comprehensive identification and expression analysis of Hsp90s gene family in Solanum lycopersicum. Genet. Mol. Res. 2015;14:7811–7820. doi: 10.4238/2015.July.14.7. PubMed DOI

Belonoznikova K., Vaverova K., Vanek T., Kolarik M., Hyskova V., Vankova R., Dobrev P., Krizek T., Hodek O., Cokrtova K., et al. Novel insights into the effect of Pythium strains on rapeseed metabolism. Microorganisms. 2020;8:1472. doi: 10.3390/microorganisms8101472. PubMed DOI PMC

Fradin E.F., Thomma B.P.H.J. Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Mol. Plant Pathol. 2006;7:71–86. doi: 10.1111/j.1364-3703.2006.00323.x. PubMed DOI

Sirima A., Sereme A., Koita K., Zida E., Nana A.T., Sereme D., Nana T., Sawadogo M. Characterization of Alternaria brassicicola isolated from tomato in Burkina Faso, and use of two essential oils for its control in vitro. Afr. J. Agric. Res. 2021;17:1371–1379. doi: 10.5897/AJAR2021.15691. DOI

Hajieghrari B., Torabi-Giglou M., Mohammadi M.R., Davari M. Biological potantial of some Iranian Trichoderma isolates in the control of soil borne plant pathogenic fungi. Afr. J. Biotechnol. 2008;7:967–972.

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Hodek O., Krizek T., Coufal P., Ryslava H. Design of experiments for amino acid extraction from tobacco leaves and their subsequent determination by capillary zone electrophoresis. Anal. Bioanal. Chem. 2017;409:2383–2391. doi: 10.1007/s00216-017-0184-2. PubMed DOI

Mittler R., Zilinskas B.A. Detection of ascorbate peroxidase-activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium. Anal. Biochem. 1993;212:540–546. doi: 10.1006/abio.1993.1366. PubMed DOI

Ricci G., Lobello M., Caccuri A.M., Galiazzo F., Federici G. Detection of glutathione transferase activity on polyacrylamide gels. Anal. Biochem. 1984;143:226–230. doi: 10.1016/0003-2697(84)90657-2. PubMed DOI

Laemmli U.K. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. PubMed DOI

Hyskova V., Pliskova V., Cerveny V., Ryslava H. NADP-dependent enzymes are involved in response to salt and hypoosmotic stress in cucumber plants. Gen. Physiol. Biophys. 2017;36:247–258. doi: 10.4149/gpb_2016053. PubMed DOI

Iven T., Konig S., Singh S., Braus-Stromeyer S.A., Bischoff M., Tietze L.F., Braus G.H., Lipka V., Feussner I., Droge-Laser W. Transcriptional activation and production of tryptophan-derived secondary metabolites in Arabidopsis roots contributes to the defense against the fungal vascular pathogen Verticillium longisporum. Mol. Plant. 2012;5:1389–1402. doi: 10.1093/mp/sss044. PubMed DOI

Pavlikova D., Zemanova V., Prochazkova D., Pavlik M., Szakova J., Wilhelmova N. The long-term effect of zinc soil contamination on selected free amino acids playing an important role in plant adaptation to stress and senescence. Ecotoxicol. Environ. Saf. 2014;100:166–170. doi: 10.1016/j.ecoenv.2013.10.028. PubMed DOI

Dulermo T., Bligny R., Gout E., Cotton P. Amino acid changes during sunflower infection by the necrotrophic fungus B. cinerea. Plant Signal. Behav. 2009;4:859–861. doi: 10.4161/psb.4.9.9397. PubMed DOI PMC

Jez J.M. Structural biology of plant sulfur metabolism: From sulfate to glutathione. J. Exp. Bot. 2019;70:4089–4103. doi: 10.1093/jxb/erz094. PubMed DOI

Zeier J. New insights into the regulation of plant immunity by amino acid metabolic pathways. Plant Cell Environ. 2013;36:2085–2103. doi: 10.1111/pce.12122. PubMed DOI

Ali S., Ganai B.A., Kamili A.N., Bhat A.A., Mir Z.A., Bhat J.A., Tyagi A., Islam S.T., Mushtaq M., Yadav P., et al. Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance. Microbiol. Res. 2018;212:29–37. doi: 10.1016/j.micres.2018.04.008. PubMed DOI

Satkova P., Stary T., Pleskova V., Zapletalova M., Kasparovsky T., Cincalova-Kubienova L., Luhova L., Mieslerova B., Mikulik J., Lochman J., et al. Diverse responses of wild and cultivated tomato to BABA, oligandrin and Oidium neolycopersici infection. Ann. Bot. 2017;119:829–840. doi: 10.1093/aob/mcw188. PubMed DOI PMC

Berka M., Kopecka R., Berkova V., Brzobohaty B., Cerny M. Regulation of heat shock proteins 70 and their role in plant immunity. J. Exp. Bot. 2022;73:1894–1909. doi: 10.1093/jxb/erab549. PubMed DOI PMC

Lee J.H., Yun H.S., Kwon C. Molecular communications between plant heat shock responses and disease resistance. Mol. Cells. 2012;34:109–116. doi: 10.1007/s10059-012-0121-3. PubMed DOI PMC

Moshe A., Gorovits R., Liu Y., Czosnek H. Tomato plant cell death induced by inhibition of HSP90 is alleviated by Tomato yellow leaf curl virus infection. Mol. Plant Pathol. 2016;17:247–260. doi: 10.1111/mpp.12275. PubMed DOI PMC

Hyskova V., Belonoznikova K., Doricova V., Kavan D., Gillarova S., Henke S., Synkova H., Ryslava H., Cerovska N. Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y. Plant Biol. 2021;23:131–141. doi: 10.1111/plb.13234. PubMed DOI

Surface properties of mycoparasitic Pythium species and their interaction with model materials