Botrytis aclada and other Botrytis spp. can cause neck rot on onions, a storage disease that normally is very difficult to detect at harvest using traditional isolation techniques. Sequence characterized amplified region primers (BA2f/BA1r) were designed based on a previously cloned and amplified DNA fragment for direct amplification of isolates of Botrytis spp. associated with neck rot of onions. Digestion of the polymerase chain reaction (PCR) amplification product with the restriction enzyme ApoI makes it possible to distinguish the five groups: Botrytis aclada types AI and AII (B. allii); B. byssoidea; B. squamosa; and B. cinerea. The detection limit was 1 to 10 pg of pure fungal DNA. It was possible to detect B. aclada with the PCR method in artificially inoculated onion bulb tissue and in mature onion leaves showing no symptoms of the disease. The availability of a sensitive and specific PCR detection and identification method for Botrytis onion neck rot pathogens should facilitate ecological studies of this group of onion pathogens.

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Fifty-one isolates representing the four Botrytis spp. associated with onion neck rot were clustered by unweighted pair group method with arithmetic mean based on universal-primed polymerase chain reaction (UP-PCR) fingerprints. Bootstrap analysis of the consensus phenogram clearly demonstrated five strong clusters among the four Botrytis spp.: B. cinerea (C), B. squamosa (S), B. byssoidea (B), and B. aclada (AI and AII). Subdivision of the 30 B. aclada isolates, AI (14) and AII (16), from Europe, Egypt, North America, and Japan was further supported by restriction analysis of the internal transcribed spacer of the ribosomal genes and spore size measurements. Gene diversities (H) among AI and AII isolates were very low (0.007 and 0.043, respectively). A likelihood ratio chi-square test (G(2)) of Nei's coefficient of genetic differentiation (G(ST)) showed that both B. aclada subgroups, AI and AII, were significantly different from B. byssoidea (P < 0.001), and that B. aclada subgroups AI and AII were significantly different from each other (P < 0.001). No UP-PCR alleles were shared by AI and B. byssoidea isolates, whereas 10 and 12 alleles were shared by AI:AII and AII:B. byssoidea, respectively. The hypothesis that AII may be a hybrid between AI and B. byssoidea is discussed.

Ulocladium atrum (isolates 385 and 302) consistently inhibited Botrytis aclada sporulation on dead onion leaf pieces under constant moist conditions and with an interrupted wetness period of 9 h. Clonostachys rosea (isolate 201) was as effective as U. atrum under constant moist conditions, but was ineffective if exposed to a drying period. No sporulation of B. aclada was observed 8 and 12 days after inoculation in the presence of U. atrum 302. C. rosea 201 significantly reduced B. aclada sporulation 8 days, but not 12 days after inoculation. When U. atrum 302 or C. rosea 201 was applied 1 day prior to B. aclada the antagonistic effect was higher compared to when the antagonists were applied on the same day. C. rosea 201 and U. atrum 302 did not obstruct the growth of B. aclada from necrotic onion leaf tips into living tissue, when artificially induced necrotic leaf tips were infested with B. aclada 24 h prior to antagonists. Three days after antagonist application, no symptoms could be observed on the healthy leaf tissue, nor was there sporulation on the necrotic leaf tip. However, B. aclada was immunologically detected 2 cm below the inoculation site. We conclude that under constant moist conditions the antagonists C. rosea 201 and U. atrum 302 cannot stop the progress of B. aclada from necrotic into fresh leaf tissue.

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