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Waiteacircinatavar. zeae,a pathogen with a relatively narrow host range, has recently been detected in cabbage and oilseed rape in Europe and worldwide. In this study, we developed specific conventional and real-time PCR protocols for direct detection of W.circinatavar. zeaefrom mycelium and diseased plant tissue. The newly developed primer pair zeaefor1/zeaerew1, used in PCR protocols, specifically amplified only target isolates of W.circinatavar. zeaewhen tested against isolates of 11 different binucleate and multinucleate anastomosis groups of Rhizoctoniaspp. including AG-A, AG-G, AG-F, AG-U, AG-2-1, AG-2-2, AG-3, AG-4 HGI, AG-4 HGII, AG-4 HGIII, and AG-6 and common soil-borne pathogens. Total of nine previously published primer pairs designed for the detection of various Rhizoctoniaspp. were also tested and did not amplify target isolates of W.circinatavar. zeae. The detection limit of conventional and real-time PCR protocols was 10–2and 10–5(with starting concentration 9.5 ng/µl), respectively, and both methods are the first available tools for direct detection and identification of W.circinatavar. zeaefrom mycelium and diseased oilseed rape seedlings. Both conventional and SYBR-Green-based real-time PCR protocols are cost-effective and provide a solid basis for further investigations of W.circinatavar. zeae, particularly in relation to distribution, host range, and epidemiology.

AbstractThe antifungal effects of thyme, cinnamon bark and clove bud essential oils (EOs) were investigated in vitroon Colletotrichum acutatummycelial growth, conidial germination, appressoria formation, and in vivoon strawberry fruit disease incidence. All tested EOs, incorporated in potato-dextrose agar, inhibitedC. acutatummycelial growth, and had a fungistatic effect at concentration 667 µl/l of medium. Volatiles of cinnamon bark, thyme and clove bud EOs completely prevented conidial germination at the lowest concentrations of 1.53, 15.3 and 76.5 µl/l of air, respectively, and disabled appressoria formation at concentration of 1.53 µl/l of air. On inoculated strawberry fruit, thyme and cinnamon bark EO volatiles reduced anthracnose incidence at concentrations above 15.3 and 76.5 µl/l of air, respectively. GC-FID and GC-MS analysis showed that major components of thyme EO were p-cymene, thymol, α-terpineol, carvacrol; cinnamon bark EO: trans- cinnameldehyde, trans-cinnamyl acetate; clove bud EO: eugenol and β-caryophyllene. Our results suggest thatvolatiles of thyme and cinnamon bark EOs are effective against C. acutatumboth in vitroand in vivo.

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5–6, with an optimum at pH 4.5. PG activity was detectable 0–70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca2+, Mg2+, Mn2+and Fe2+and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.