Etiology of blue mold of pome fruits in Serbia and physiological changes in infected apple fruit

[A. A. Žebeljan]

127 str.

Biotehničke nauke - Fitopatologija / Biotechnology - Phytopathology

Plava trulež koju prouzrokuju vrste iz roda Penicillium jedna je od najznačajnijih bolesti jabučastog voća u toku čuvanja. Iz skladišta sa različitim uslovima čuvanja iz 20 lokaliteta u Srbiji tokom 2014. i 2015. godine, prikupljeni su plodovi jabuke, kruške, dunje i mušmule sa simptomima plave truleži. Simptomi su varirali od sitnih, svetlo smeđih, vodenastih pega do potpune truleži plodova. Na zaraženim plodovima su u većini slučajeva bile prisutne plavozelene spore. Iz prikupljenih plodova izolovane su gljive koje su po morfološkim karakteristikama odgovarale Penicillium spp. Patogenost izolata je proverena veštačkim inokulacijama plodova jabuke i voća iz kog su izolovane. Dobijeni izolati (ukupno 96) molekularno su identifikovani, a odabrani izolati (ukupno 26) su okarakterisani na osnovu ITS, BenA, CaM i RPB2 regiona, kao i pojedinačnih i multolokus filogenetskih analiza. Izolati su makroskopski i mikroskopski morfološki okarakterisani na diferencijalnim podlogama MEA, CYA, YES i PDA i ispitana je njihova virulentnost na plodovima jabuka sorti Zlatni Delišes, Crveni Delišes i Greni Smit. U plodovima jabuke sorte Ajdared inokulisanim izolatima P. expansum i P. crustosum analizirane su fiziološke promene u primarnim metabolitima i antioksidantima, a njihova dinamika je praćena od momenta inokulacije do razvoja bolesti (11 dpi). Kao prouzrokovači plave truleži jabučastog voća u Srbiji identifikovane su tri vrste roda Penicillium: P. expansum, P. crustosum i P. solitum. Najčešće izolovana vrsta bila je P. expansum (91,67%), a zatim P. crustosum (7,29%) i P. solitum (1,04%). P. expansum je izolovan iz plodova jabuke, kruške i dunje, P. crustosum iz plodova jabuke, kruške, dunje i mušmule, a P. solitum iz ploda dunje. U okviru ovih istraživanja su kao prouzrokovači plave truleži prvi put u svetu opisane P. crustosum i P. solitum na plodovima dunje i P. crustosum na plodovima mušmule, dok je u Srbiji prvi put opisana P. expansum na plodovima dunje. Na osnovu molekularnih karakteristika i filogenetskih analiza jasno su se razdvojile vrste P. expansum, P. crustosum i P. solitum. U okviru vrsta P. expansum i P. crustosum uočena je genetička varijabilnost (u tri, odnosno dva lokusa) i opisani su haplotipovi. Izolati sve tri vrste formirali su: plavozelene kolonije sa ili bez eksudata, različitih boja naličja, terverticillata, ređe biverticillata konidiofore, cilindrične rame i metule, cilindrične do ampuliformne fijalide i konidije glatkih zidova u nizu. Konidiofore P. expansum bile su glatkih zidova, P. crustosum nazubljenih, dok su konidiofore P. solitum bile glatke ili blago nazubljenih zidova. P. solitum je formirao najveće konidije. Na inokulisanim plodovima različitih sorti jabuke, P. expansum je ispoljio veću virulentnost od P. crustosum, a P. solitum je ispoljio najslabiju virulentnost. P. expansum je bio najvirulentniji na plodovima sorte jabuke Crveni Delišes, dok je P. crustosum bio podjednako virulentan na plodovima jabuka sorti Zlatni Delišes i Crveni Delišes. Obe vrste su najslabiju virulentnost ispoljile na plodovima jabuke sorte Greni Smit. Najveća razlika u virulentnosti između P. expansum i P. crustosum uočena je na plodovima jabuke sorte Crveni Delišes. Na poprečnom preseku inokulisanih plodova jabuke P. crustosum je izazvao trulež tamnije boje i sporulacija je bila prisutna i u unutrašnjosti plodova, za razliku od P. expansum koji je prouzrokovao svetlo smeđu trulež, a sporulacija je bila prisutna samo oko mesta inokulacije. PCA i klaster analize su na osnovu molekularnih odlika, morfoloških karakteristika i virulentnosti jasno razdvojile vrste i potvrdile njihovu identifikaciju. Takođe, u okviru P. expansum i P. crustosum potvrđena je intraspecijska varijabilnost. Izdvojile su se dve grupe u okviru P. expansum na osnovu brzine porasta kolonije, boje naličja kolonija na PDA i MEA (krem ili žuto naličje), razlike u obojenosti naličja na CYA (krem, žuto, krem roze do crveno naličje) i filogenije (BenA, CaM, RPB2 i multilokus). U okviru P. crustosum izdvojile su se tri grupe na osnovu brzine porasta kolonija, filogenije (RPB2 i multilokus) i virulentnosti. U plodovima jabuke sorte Ajdared nakon inokulacije izolatima P. expansum i P. crustosum došlo je do aktiviranja odbrambenih mehanizama...

Blue mold, caused by Penicillium species, is one of the most important postharvest diseases of pome fruit. During 2014 and 2015, from 20 storage facilities in Serbia, apple, pear, quince and medlar fruits with blue mold symptoms were collected. Symptoms ranged from small, light brown, soft, watery lesions to complete decay, and in most cases blue-green colored spores were present. Fungi with morphological characteristics of Penicillium spp. were isolated and their pathogenicity was tested. Obtained isolates (96 in total) were identified and selected isolates (26 in total) were characterized based on single and multilocus phylogenetic analysis of ITS, BenA, CaM and RPB2. The isolates were morphologically characterized on differential media MEA, CYA, YES and PDA and their virulence was tested on apple fruit cvs. Golden Delicious, Red Delicious and Granny Smith. In apple fruit cv. Idared infected with P. expansum and P. crustosum physiological changes of primary metabolites and antioxidants were evaluated. Three species of the genus Penicillium have been identified as the causal agents of blue mold of pome fruit in Serbia: P. expansum, P. crustosum and P. solitum. The most frequently isolated species was P. expansum (91.67%), followed by P. crustosum (7.29%), and P. solitum (1.04%). P. expansum was isolated from apple, pear and quince fruit, P. crustosum from apple, pear, quince and medlar fruit, and P. solitum from quince fruit. In this study, P. crustosum and P. solitum were described for the first time as causal agents of blue mold of quince fruit in the world while P. crustosum on medlar and P. expansum on quince fruit are novel findings in Serbia. P. expansum, P. crustosum and P. solitum were clearly separated based on molecular characteristics and phylogeny. Within P. expansum and P. crustosum genetic variability was observed in three and two loci, respectively, indicating presence of different haplotypes. Morphologically, all isolates formed blue-green colonies with or without exudate and different reverse color. Conidiophores were terverticillata, occasionally biverticillata, rami and metulae were cylindrical, phialides were cylindrical to ampuliform, and conidia were smooth-walled and formed in chains. Conidiophores of P. expansum were smooth-walled, of P. crustosum with roughened walls, and of P. solitum with smooth or finely roughened walls. P. solitum had the largest conidia. P. expansum was more virulent than P. crustosum, while P. solitum was the least virulent on inoculated apple fruit of all tested cultivars. P. expansum was the most virulent on cv. Red Delicious and produced larger lesions than on cv. Golden Delicious. P. crustosum was equally virulent on cvs. Red Delicious and Golden Delicious. Both species were the least virulent on cv. Granny Smith. The largest difference in virulence between P. expansum and P. crustosum was observed on cv. Red Delicious. On the cross section of inoculated apple fruit differences between the decay symptoms caused by P. expansum and P. crustosum were observed. P. crustosum isolates caused slightly darker color of the decayed tissue in all cultivars and sporulated both around the wound an in the apple flesh of cv. Red Delicious. PCA and cluster analysis based on the molecular characteristics, morphological characteristics and virulence separated the species and confirmed the identification. Also, in P. expansum and P. crustosum intraspecific variability was proven. Two groups were separated in P. expansum which differed in colony growth, reverse color on PDA and MEA (cream or yellow reverse), CYA reverse (cream, yellow, salmon pink to red), and phylogeny (BenA, CaM, RPB2 and multilocus). Within P. crustosum three groups were separated based on colony growth, phylogeny (RPB2 and multilocus) and virulence. Temporal changes in defense mechanisms of apple fruit cv. Idared were studied following inoculation with isolates of P. expansum and P. crustosum by analysing the concentrations of primary metabolites (sugars: sucrose, glucose and fructose and organic acids: malic and shikimic acids) and antioxidants (glutathione, ascorbate and phenol)..

Penicillium expansum, P. crustosum, P. solitum, plodovi jabuke, kruške, dunje i mušmule, filogenija, morfologija, virulentnost, intraspecijska varijabilnost, askorbatno-glutationski ciklus, fenoli, primarni metaboliti

Penicillium expansum, P. crustosum, P. solitum, apple, pear, quince and medlar fruits, phylogeny, morphology, virulence, intraspecies variability, ascorbate-glutathione system, phenolics, primary metabolites

632.4:634.12(497.11)(043.3)

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