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ItemDetection and characterization of virulent, hypovirulent, and nonvirulent Clavibacter Michiganensis subsp. Michiganensis(University of Hawaii at Manoa, 2003-08)Clavibacter michiganensis subsp. michiganensis (Cmm), a seedborne quarantine pathogen, causes bacterial canker of tomato, which is a serious disease that can severely decrease yields in greenhouse and field production areas. Seed assays are used to prevent dissemination of Cmm through infested seed, but limitations in assay sensitivity and specificity allow canker outbreaks to continue. An assay was developed that detected Cmm when as few as 10 colony forming units (cfu) were present per 50 ml sample. The assay used a three-unit filtration system to capture bacterial cells, followed by a four-day membrane incubation and a colony blot immunoassay using the CMMI monoclonal antibody (MAb). The filtration and immunoassay technique was more sensitive than a standard spread plating assay, and could potentially reduce current assay times by up to 3 weeks. Assays done on nine seed lots yielded a high percentage (81%) of MAb CMMl positive colonies that were hypovirulent or nonvirulent on tomato (Lycopersicon esculentum cv. Kewalo). All strains were confirmed as Cmm using the MicroLog identification system, rep-PCR, two PCR primer sets, and an endoglucanase assay. Of the assays tested, MicroLog and rep-PCR were the most consistent in identifying hypovirulent and nonvirulent MAb CMMI-positive strains as Cmm. The CMMI MAb was further used to quantify in planta movement and multiplication of a nonvirulent Cmm strain when coinoculated with a virulent Cmm strain. In planta coincubation did not significantly alter growth or colonization habits of either strain. Thus, here is no evidence that nonvirulent Cmm strains playa role in bacterial canker epidemiology. However, their continued isolation from diseased and infested tissues, and the importance of nonvirulent strains in other pathosystems, suggest significance and warrants further investigation.
ItemComparison of Three Different Esterase Phenotype Isolates of the Kona Coffee Root-Knot Nematode, Meliodogyne Konaensis(University of Hawaii at Manoa, 2002-12)The esterase phenotype of female Meloidogyne konaensis (MK) was originally described as a single fast band (F1). Culturing MK, originally isolated from coffee, on tomato resulted in the detection of two additional phenotypes (I1-FI and I1). These three M. konaensis esterase phenotypes (MKF1, MKl1-FI and MKI1) were evaluated for molecular and morphological behavior. The isolate MKI1-F1 exhibited two esterase bands (F1 and I1). The MKI1 exhibited a slow migrating band identical to that of M. incognita. All three isolates had an MDH phenotype N1, identical to that of M. javanica and M. incognita. On a 2-dimensional electrophoresis gel (2-DGE), the protein maps of all three isolates differed from that of M. javanica. There were similar protein spots between isolates of M. konaensis. Morphological comparison showed differences in the male heads and perineal patterns among three isolates. All MKF1 males had the typical high and rounded head shape and stylet shaft with protuberance. MKl1-FI males also had the rounded head shape but the majority of head caps were lower than those of MKF1. The majority of MKI1 male heads caps was narrow, square and indented anteriorly. Host range and pathogenicity differed among the three isolates. MKF1 had low reproduction on coffee, whereas MKI1-F1 and MKI1 failed to develop on coffee. The reproduction ratio on coffee by MKF1 was 1.1, and zero for MKl1-F1 and MKl1. In contrast, reproduction was high on tomato and cucumber. MKl1-F1 had the highest reproduction on tomato and cucumber which was two times more than MKF1 and MKl1, whereas MKF1 and MKl1 were similar. The reproduction ratio on tomato and cucumber was 110.9 and 40.8 by MKl1-F1, 45.9 and 9.3 by MKF1, and 47.6 and 17.6 by MKI1 respectively. Overall, MKF1 was the only isolate having the ability to infest coffee and MKI1-F1 was the most aggressive isolate of M. konaensis on tomato and cucumber.