CC BY
0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
CHITOSAN AND ARBUSCULAR MYCORRHIZAE AS PRIMING AGENTS AGAINST PLASMOPARA VITICOLA IN
GRAPEVINES
Marco Monticelli
PhD student University of Camerino https://doi.org/10.5281/zenodo.13827436
Keywords: chitosan, arbuscular mycorrhizae, plant priming, Plasmopara viticola.
Following the recent trends in organic agriculture and as a response to climate change, this review aims at presenting two alternative methods to control Plasmopara viticola in Vitis vinifera. The first involves chitosan, which can be delivered both as a foliar spray to protect and prime plants for an incoming fungal sporulation after a rainfall, and as soil amendment to stimulate arbuscular mycorrhizae (AM) colonization. Mycorrhization, the second overviewed method, on turn primes plants, but in a prolonged way, able to confer a more resistant physiological state, alongside increasing plant productivity. Priming by chitosan or AM enhances defence genes transcription, like chitinase, phytoalexins and phenylpropanoids in the host, all of which are either antioxidant or toxic compounds for pathogenic fungal microorganisms.
Grapevine is a common plant whose fruit are edible or fermented to produce wine, mainly present in temperate, especially mediterranean, climatic zones and currently is the most remunerative fruit crop, with a global market size of over 29 billion euros. However, domesticated strains have become highly susceptible to fungal infections, like Botrytis cinerea, responsible for grey mould, but more notably the American biotrophic oomycete Plasmopara viticola, grape downy mildew causing agent, capable of yield loss up to 75% [16]. Copper and copper mixtures are by far the most utilized fungicides for P. viticola, but studies demonstrated that its accumulation has have negative effects on soil organisms, plant roots and surrounding ecosystem [22]. For this reasons research of alternatives is encouraged by European Union. Among the reviewed strategies there are elicitor compounds, those capable of triggering a mild immune response in the plant, allowing to recall more effectively the induced defence mechanism on successive exposures to the pathogen [8], in a vaccine-like fashion (also synthetic compounds can prime plants, but they will not be reviewed here). Upon pathogen attack, primed plants show a faster and stronger oxidative burst, defence genes upregulation and phytoalexins accumulation, compared to non-primed plants [15].
Chitin, one of the most naturally abundant compounds on earth, is a linear polymer of N-acetyl-D-glucosamine units linked by P-(1—>4) linkages, and chitosan is obtained by at least 70% deacetylation of chitin. Chitin and chitosan have the same overall function, but in biotechnology chitosan is preferred as it possesses unique amino groups that make it soluble in water, while chitin is not [19]. Chitosan proved to be a reliable compound for fungal diseases control [2], by being diluted and sprayed directly on the interested fruit or vegetable (post-harvest) and/or on the aerial part of the plant colonizable by the pathogen (pre-harvest). It has a wide protection spectrum, it is not known to harm animals, plants or microorganisms and it is biodegradable. When sprayed on leaves, it forms a film that acts as (1) a mechanical barrier, (2) an antimicrobial compound, and (3) an elicitor of defence mechanisms in the host. However, the coating formed on leaves also causes stomatal occlusion resulting in decreased vegetative growth, though with unchanged grape
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
production [17]. Among these features, the eliciting activity of chitosan is due to its chemical structure, a pathogen associated molecular pattern, recognized by the plant as a component of fungi cell wall or insect exoskeleton. Then the plant enters a defence state that stimulates production of pathogenesis related proteins such as chitinase, P-1,3 glucanase and ammonia lyase [6]. For what regards V. vinifera protection from downy mildew and grey mould, it has been observed in leaves an accumulation of piceid, resveratrol and s-viniferin, when chitosan had been sprayed within 48h of the pathogen inoculation. These defence proteins are toxic for fungi and resulted in a reduction of P. viticola mycelia spreading from 40% (control) to 10% of leaf area, and from 20% to 10% in botrytis [1].
Among the proposed replacement for copper-based treatments to downy mildew, arbuscular mycorrhizae are mentioned as soil fungal elicitors of priming, that differently from chitosan, establish a long-term symbiosis and hence resistance to pathogens. Moreover, they promote nutrient uptake and abiotic stress alleviation, for example to drought conditions, salts imbalance or nutrient deficiency [3]. Two studies reported promising results in mycorrhized grapevines, describing an increase of antioxidant compounds in plant leaves upon P. viticola and B. cinerea inoculation, [4] and decrease of P. viticola effectors, respect to non-mycorrhized vines [7]. Induction of mycorrhization though inoculum seeding could reduce costs in agriculture due to rarefaction of treatments because once established the symbiosis endures if tillage and herbicides are not applied [23,12]. Furthermore, mycorrhization and phosphorous fertilization demonstrated to reduce copper toxicity in vineyard soils [5]. Interestingly, a study showed that the class of stilbenoid compounds produced by a hybrid strain of V. vinifera constitutively resistant to P. Viticola, was the same stimulated by mycorrhizal and chitosan priming in non-resistant plants [14]. As a further confirm, not just the pathogenesis related proteins are the same in the primed and resistant vines but also the expression of the enzymes which synthesize them, namely stilbene synthase and phenylalanine ammonia lyase, as it can be compared among the previous and next study [13]. For what regards the most suited AM species for inoculum seeding, application of more than one species has shown better results than inoculation of a single variety. Anyway, most of the studies were conducted with the Glomeraceae family, notably the Glomus and Rhizophagus species.
About the combination of chitosan and AM, [11] reported positive effects boosted mycorrhization, enhanced length of extra radical mycelium and number of spores of G. mossae, while [10] noted increased root and shoot growth, chlorophyll fluorescence and xylem vessels diameters of plant. Conversely, other studies reported no or decreased mycorrhization in mais when chitosan and AM inoculum were added to soil [20], and in tomato when chitosan was either sprayed or drenched [9]. The authors inferred that the mineral cations present in nutrient rich soil could bind and block chitosan, and that chitinases produced upon foliar chitosan exposure can react with chitin in the fungal cell wall of mycorrhizae. Therefore, the feasibility of achieving plant protection through combination of chitosan application and mycorrhizal fostering should be better tested in grapevines. Finally, the aromatic profile of wine is not altered by the chitosan treatment [17], rather some authors reported better qualities of berries in terms of sugars, phenols and anthocyanin content in mycorrhized grapevines [21].
Summarizing, to reduce P. viticola pathogenicity by limiting copper abuse, chitosan and AM represent respectively a short-term and long-term organic and potentially cheap solutions. Effects of these treatments on untargeted organisms and local biodiversity should also be
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
evaluated. Chitosan has already been commercialized in organic agriculture under EU legislation, even though it has not yet been widely adopted partly because, despite being a waste product of the sea food, fungi and insect food chains, the retail price is at least double that of copper. This problem is already being addressed with new techniques of chitin extraction and chitosan. Likewise, producing inoculum of AM at industrial scales is not efficient due to the low density of propagules in the bulk material. Still, nutrient film techniques, root organic culture and aeroponics are promising production techniques. A final remark on chitosan treatments, is that with chitin deriving from crustaceans or insects, the grapes or wine could not be classified as vegan. To conclude, each ecosystem, farm and climate condition requires tailored solutions for the individual or combined application of chitosan and arbuscular mycorrhizae to prevent fungal diseases in grapevines and other crops.
REFERENCES:
1. Aziz A. Chitosan Oligomers and Copper Sulfate Induce Grapevine Defense Reactions and Resistance to GrayMold and Downy Mildew. "Phytopathology" 2006 - 96(11):1188-94
2. Bautista-Baños S. Chitosan as a potential natural compound to control pre and postharvest diseases of horticultural commodities. "Crop Prot" 2006 - 25 (2), pp. 108-118
3. Bonfante P. Mechanisms underlying beneficial plant -fungus interactions in mycorrhizal symbiosis. "Nat Commun" 2010 - 1, 48
4. Bruisson S. Arbuscular mycorrhizal symbiosis stimulates key genes of the phenylpropanoid biosynthesis and stilbenoid production in grapevine leaves in response to downy mildew and grey mould infection. "Phytochemistry" 2016
5. Brunetto G. Use of phosphorus fertilization and mycorrhization as strategies for reducing copper toxicity in young grapevines. "Scientia Horticulturae" 2019 - Volume 248, Pages 176183
6. Buonaurio R. Induced resistance to plant diseases caused by Oomycetes and fungi. "Petria" 2009 - 19(3), 130-148.
7. Cruz-Silva A. First Insights into the Effect of Mycorrhizae on the Expression of Pathogen Effectors during the Infection of Grapevine with Plasmopara viticola. "Sustainability" 2021 -13. 1226.
8. Dagostin S. Are there alternatives to copper for controlling grapevine downy mildew in organic viticulture? "Crop Protection" 2011 - Vol 30, Iss 7, Pag 776-788
9. El Amerany F. Foliar Application of Chitosan Increases Tomato Growth and Influences Mycorrhization and Expression of Endochitinase-Encoding Genes. "Int. J. Mol. Sci." 2020 -21, 535
10. El Amerany F. The effect of chitosan, arbuscular mycorrhizal fungi, and compost applied individually or in combination on growth, nutrient uptake, and stem anatomy of tomato. "Scientia Horticulturae" 2020 - 261
11. Gryndler M. Chitin stimulates development and sporulation of arbuscular mycorrhizal fungi. "Applied Soil Ecology" 2003 - Vol. 22, Iss. 3, Pag. 283-287
12. Jasper D.A. Soil disturbance reduces the infectivity of external hyphae of vesicular-arbuscular mycorrhizal fungi. "New Phytol" 1989 - 112, pp. 93-99
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS"
_25-26 SEPTEMBER, 2024_
13. Kortekamp A. Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen. "Plant Physiology and Biochemistry" 2006 -Volume 44, Issue 1, Pages 58-67
14. Malacarne G. Resistance to Plasmopara viticola in a grapevine segregating population is associated with stilbenoid accumulation and with specific host transcriptional responses. "BMC Plant Biol." 2011 - 11:114
15. Martinez-Medina A. Recognizing Plant Defense Priming. "Trends in Plant Science" 2016 -1455
16. Organization of Vine and Wine 2017 OIV Statistical Report on World Vitiviniculture.
17. Romanazzi G. Chitosan, a biopolymer with triple action on postharvest decay of fruit and vegetables: eliciting, antimicrobial and film-forming properties. "Frontiers in Microbiology" 2018
18. Romanazzi G. Impact of alternative fungicides on grape downy mildew control and vine growth and development. "Plant Disease" 2016
19. Shahidi F. Chemistry, Production, Applications, and Health Effects. "Advances in Food and Nutrition Research" 2005 - Volume 49, Pages 93-135
20. Singh N. Effect of chitosan on plant growth and mycorrhization in Zea mays. "Journal of Mycopathological Research" 2021 - 59. 291-294.
21. Torres N. Arbuscular Mycorrhizal Symbiosis as a Promising Resource for Improving Berry Quality in Grapevines Under Changing Environments. "Front Plant Sci" 2018 - 9:897
22. Yu Z. Antimicrobial resistance and its association with tolerance to heavy metals in agriculture production. "Food Microbiology" 2017 - 64:23-32
23. Zaller J.G. Herbicides in vineyards reduce grapevine root mycorrhization and alter soil microorganisms and the nutrient composition in grapevine roots, leaves, xylem sap and grape juice. "Environ Sci Pollut Res" 2018 - 25
