Ph.D. - Botanical Sciences (Plant Pathology)

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    Genetics of Choanephora cucurbitarum
    ( 1996) Yu, Ming-qiu
    Zygospores of Choanephora cucurbitarum (Berkeley et Ravevel) Thaxter germinated by producing germ tubes with terminal germsporangia, germsporangioles on vesicles or germ tubes which continued to grow without producing germsporangia. Zygospores have a light requirement for germination and were stimulated by KMnO4, NaClO and H2O2. Zygospore progeny from crosses between (+) and (-) isolates of g. cucurbitarum consisted of (+), (-) and (+,-) mating types. Mating types of germsporangiospores in each germsporangium were either all (+), or all (-) from respective (+) or (-) zygospore cultures and, all (+,-), or (+) and/or (-) plus (+,-) types from (+,-) zygospore cultures. Mating type heterokaryons (+,-) obtained from zygospore and germsporangiospore cultures in all crosses produced azygospores and zygospores which segregated into (+) and (-) during asexual propagation. All the zygospore and germsporangiospore progenies from the cross between two fast growing isolates were of fast growing type, while all those from the crosses between two slow growing isolates were of slow growing type. The crosses between fast and slow growing isolates gave rise to progeny with both fast and slow growing types. Mating types of zygospore isolates from crosses between two near identical isolates followed 1(+):1(-):2(+,-) ratio. The distribution of mating types (+) and (-), and growth types F and S fit a 1:1 ratio in zygospore progeny in all crosses. Results suggest that mating type as well as growth type are each controlled by a gene with a pair of alleles, and that these two genes are linked to each other with a distance of about 33 cM. The heterokaryotic zygospore progeny from the crosses between F and S types consisted of F(+,-), S(+,-), F,S(+,-), S,F(+,-), F,S(+) and F,S(-). Each of them segregated into two types during sporangiospore formation, suggesting that only two of the four nuclei resulted from meiosis move into the germ tube of a germinating zygospore. Mating type heterokaryons (+,-) formed through fusion of (+) and (-) protoplasts. The (+,-) colonies from protoplast fusion also produced azygospores and zygospores which segregated into (+) and (-) mating types during sporangiospore formation.
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    Differential epidemiological fitness among strains of Xanthomonas campestris pv. campestris and the genetics of pathogenicity
    ( 1996) Shigaki, Toshiro
    Variation among strains of Xanthomonas campestris pv. campestris, the causal organism of black rot of crucifers, was studied. The relationship between symptoms associated with selected strains, their epidemiological fitness in misted seedbeds, the presence of a gene associated with severe symptoms in these strains, and a pathovar-specific surface antigen was examined. Strains with blight induction capacity spread more rapidly in misted seedbeds. These blight strains all hybridized with a 5.4-kb DNA fragment isolated from the type strain of X. c. campestris (Xcc528T ). This fragment in pJC41 was previously reported to confer blight symptom induction in cabbage when mobilized to a closely related leaf spot pathogen X. c. armoraciae. The relationship between this 5.4-kb DNA fragment and blight symptom induction was studied by marker-exchange mutagenesis of Xcc528T at the pJC41 region. Loss of the fragment did not destroy the systemicity and blight induction capability of the wild type. However, in pathogenicity studies strains of X. c. campestris that hybridized with pJC41 generally caused strong blight in cabbage. Thus, it is likely that other gene(s) with similar functions exist on the genome. A positive reaction to the monoclonal antibody (MAb) All was associated with strains that produced blight. The hypothesis that the antigen reactivity with this MAb plays a role in blight induction was tested by mutagenizing a blight strain of X. c. campestris (CAMI9) at a gene locus that is necessary for the production of this antigen. Although the two A11-negative mutants (NTG901 and NTG2230) which were obtained were avirulent or had reduced virulence, the restoration of antigenicity by complementation did not result in simultaneous restoration of virulence. Thus, although the antigen for MAb All is a useful marker associated with blight-causing strains, it does not play a role in blight symptom induction or pathogenicity. In summary, blight strains of X. c. campestris are distinct from the more typical black rot strains in symptom induction and epidemiological capability. They usually, but not always, hybridize with pJC41 and react with MAb A11. It is suspected that blight induction is controlled by multiple genes.
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    Substances of plant and fungal origins stimulatory to sexual reproduction in Phytophthora
    ( 1995) Jee, Hyeong Jin
    Stimulatory effect of phospholipids, lecithin and cephalin on sexual reproduction of Phytophthora cactorum was increased by purification with column chromatography or thin layer chromatography (TLC). Gas chromatography (GC) and mass spectrometry analysis (MS) failed to detect sterols in the highly purified phospholipid samples. Trace amount of cholesterol added to the cephalin did not significantly changed the amount of oospores produced. Therefore, confirming the previous reports that sterols are not essential to sexual reproduction in pythiaceous fungi. Purification and identification of oospore stimulatory compounds from com oil and crude soybean lecithin were carried out by saponification, Florisil column chromatography, TLC, digitonin precipitation, high performance liquid chromatography and GC-MS analysis. Phytol, one of the compound identified from com oil, stimulated sexual reproduction of P. cactorum and P. parasitica. The minimum concentrations of phytol stimulatory to sexual reproduction of P. cactorum and P. parasitica were 0.002 ppm. Lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic acids, and geraniol and squalene induced oospore formation of P. cactorum following purification on TLC. Purified palmitoleic acid was stimulatory to oospore formation of P. cactorum and P. parasitica. Retinol and its esters were also stimulatory to sexual reproduction of P. cactorum and P. parasitica. Nutrient deprivation or growth limitation induced sexual reproduction of P. cactorum cultured on basal medium. The fungus grown in solid or liquid basal medium formed oospores after being transferred to water agarose. Mycelial age, concentration of the basal medium and size of petri plate affected the number of oospores produced by P. cactorum. The non-saponifiables extracted from mycelia of P. cactorum cultured in liquid basal medium stimulated oospore formation of P. cactorum and P. parasitica, while the saponifiables were stimulatory to P. cactorum only. Over all results showed that many compounds commonly found in plants and fungi are stimulatory to sexual reproduction in pythiaceous fungi. The contention that this group of fungi has an absolute requirement of exogenous sterols is, therefore, refuted.
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    Serological and pathological evaluations of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice
    ( 1995) Rehman, Faiz-Ur
    Six new serological groups IIb, VI, V, VII, VIII and IV were delineated with new monoclonal antibodies (MAbs) Xoo-7, Xoo-8, Xoo-9, Xoo-10 and Xoo-11 made to Asian strains and tested against 299 strains of Xanthomonas oryzae pv. oryzae representing various rice growing areas of the world. Serologically distinct populations were recognized by new MAbs among Indian and Nepalese strains of X o. oryzae. No relationship between serogroups and virulence or serogroup and race was obtained, except that previously reported serogroups III, IV and V contained only weakly virulent, atypical strains of X o. oryzae from the United States. Ninety seven percent of the typical X o. oryzae strains reacted either with MAb Xoo-7 or a previously reported MAb, Xco-2, but no strain reacted with both. These MAbs identified lipopolysaccharide antigens and gave bright fluorescence In indirect immunofluorescence microscopy (IF). An immunofluorescence colony staining technique (IFC) also was developed for detection of X. o. oryzae from rice seed extracts with fluorescein isothyocyanate (FITC) conjugated MAbs Xoo-2 and Xoo-7 after enrichment in a semiselective medium E. Colonies of typical strains of X. o. oryzae on medium E formed 3 to 4 days earlier than on other semiselective media. IFC enabled detection of as few as 44 cfu in 100 µl extract from 100 rice seeds (1% infestation rate) even when plates were crowded with contaminants. Since both antibodies give bright immunofluorescence, a mixture of these MAbs can be used with IFC to detect 97% of the typical strains of X. o. oryzae. Enrichment on medium E followed by identification of X. o. oryzae by IFC with two pathovar specific MAbs enhanced sensitivity of detection, and the method is useful for epidemiological and seed transmission studies of this pathogen.
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    Pathogenic associations with yellows disease of Dodonaea viscosa in Hawaiʻi
    ( 1992) Borth, Wayne B.
    Dodonaea viscosa (L.) Jacq. in Hawai'i is afflicted with a severe yellowing disorder with symptoms which include the production of pendulous, chlorotic witches' brooms, a decrease in leaf size combined with the distortion of leaf lamina, the suppression of flowering, and progressive defoliation leading to the eventual death of afflicted plants. The disease was first reported in 1984 occurring in Hawai'i Volcanoes National Park on the island of Hawai'i and has since been observed on the islands of Kauai, Maui, and Oahu. Field studies initiated in 1988 and continued over a three year period indicate a slow rate of spread of the disease based upon visual symptom expression. Viruslike particles 16 nm in diameter and 700 nm in length and double-stranded RNA of molecular weight 3 X 106 daltons were isolated from diseased plants but were absent from healthy plants. DNA complementary to dsRNA was cloned in E. coli and shown to be of non-host origin. Leaves from symptomatic and symptomless plants collected from field sites on Hawai'i, Maui, and Kauai were tested with a probe prepared from the highly conserved 165 ribosomal gene of Western X MLO, which has been shown to reliably detect MLOs from a wide range of hosts. On all the islands sampled, 80% of the symptomatic plants and 33% of the symptomless plants growing near diseased plants tested positively with this probe. Leaves and roots of healthy plants grown from seed collected from symptomless D. viscosa did not react with this probe. Pleiomorphic bodies resembling MLOs were observed in phloem tissues of diseased plants using DAPI staining and transmission electron microscopy. Such structures were not observed in healthy plants grown from seed. Partial alleviation of symptoms on diseased plants was noted following stem injections of oxytetracycline at 100 µg/ml. Attempts to transmit MLOs between D. viscosa and Catharanthus roseus by Cuscuta sandwichiana and C. campestris were not successful. The evidence suggests a complex etiology for the yellows disease of Dodonaea viscosa in Hawai'i which includes both viral and mycoplasmal agents.
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    Bacterial bioluminescence: a tool to study host-pathogen interactions between Brassica oleracea and the bacterial phytopathogen Xanthomonas campestris pv. campestris in black rot of cabbage
    ( 1991) McElhaney, Rosemarie
    Black rot of cabbage, caused by the bacterial pathogen Xanthomonas campestris pv, campestris, is a very serious disease, which results in significant crop losses worldwide. Gene technology has provided a unique marker, bacterial bioluminescence, to trace the movement of bacteria in an infected plant and their spread in the field without interrupting the disease process. Xanthomonas campestris pv. campestris strain G-171 was tranformed to the bioluminescent strain 171LIIH-7 in a mating with Escherichia coli HB101 harboringplasmidpUCD607. The plasmid carries the genes for bioluminescence and four antibiotic resistance loci. Stability of the plasmid-borne genes as well as expression of pathogenicity and virulence were confirmed in serial passage through agar plates and cabbage seedlings. Southern hybridization patterns and other in vitro tests suggest that the genes for bioluminescence and antibiotic resistances are integrated into the chromosome. Growth of the transconjugant at different temperatures and with various nitrogen compounds was compared to that of the wild type strain in liquid culture, and showed that the two strains behave similarly except for a temperature sensitivity for 171LIIH-7 above 30 C. Once it was determined that the transconjugant stably expressed bioluminescence and pathogenicity, the applicability of the bioluminescence marker to studies of black rot of cabbage was examined. The effect of host nutrition on disease progression and severity was investigated by growing Brassicaoleracea seedlingswith varying amounts of different nitrogen sources, potassium, and phosphorus. In situ movement of the bacteria in infected seedlings was monitored with X-ray film. It was determined that insufficient amounts of nitrogen increased disease, but that amounts above those required for optimum plant growth reduced disease. No significant effects on disease severity were observed from varying the amounts of potassium and phosphorus fertilization; however, when phosphorus was omitted, disease development was greatly inhibited. The transconjugant was also used to confirm disease transmission via roots and to examine transmission by whiteflies. The bioluminescent pathogen was transmitted through the roots in one out of ten seedlings, which were grown in potting mix containing black rot infected cabbage leaves. The pathogen was traced from a location in the root system into the leaves with X-ray film. The role of whiteflies in the spread of black rot of cabbage was not determined in this study, because white flies did not transmit the pathogen to healthy seedlings.
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    Genetics of Phytophthora : evidence for hybridization
    ( 1990) Chang, Tun-tschu
    Single-zoospore cultures from two isolates (A1 and A2) of Phytophthora infestans after metalaxyl treatment consisted of A1 and A2. Metalaxyl also caused mating type change in 3 A1 of g. parasitica. The conversion of A1 to A2 by metalaxyl is postulated as a possible origin of A2 of g. infestans in Europe. When g. parasitica was treated with metalaxyl for 6 weeks, many of its zoospore progeny became resistant to metalaxyl. Exposure to metalaxyl also caused zoospore progeny to change colony morphology, growth rate, ability to produce sporangia and ability of zoospores to form colonies, indicating that metalaxyl is mutagenic. The optimum conditions for activation of oospores of g. infestans were to treat oospores with 0.25% KMn04 for 15 min. Light was required for oospore germination during germination but not during maturation. Isozyme patterns of E. infestans show that selfed progeny from isolates heterozygous at PEP and GPI-1 loci segregated 1:2:1, and that all selfed progeny from homozygous isolates were identical with their respective parents indicating that £. infestans is diploid in vegetative state. All sexual progeny from the cross 903SrX947Cpr were resistant to either chloramphenicol or streptomycin and were hybrid at PEP locus suggesting that streptomycin-resistance and chloramphenicol-resistance genes were located in cytoplasm. Selfed progeny from metalaxyl-resistant (Mr) mutants of g. parasitica segregated 3 resistant : 1 sensitive. Selfed progeny from chloroneb-resistant (Cnr) mutants also segregated 3 resistant : 1 sensitive, indicating that metalaxyl and chloroneb resistance in these mutants are each conferred by a single dominant gene in heterozygous condition. Progeny from the pairing between homozygous Mr and wild type consisted of selfs and hybrids. Progeny from the pairing between homozygous Cnr resistant to chloramphenicol and resistant to streptomycin consisted of hybrids resistant to either chloramphenicol or streptomycin, suggesting that chloramphenicol-resistance and streptomycin-resistance genes are present in cytoplasm. Progeny from the pairing between homozygous Mr, Cpr A1 and homozygous Cnr, Sr A2 consisted of 4 selfs from Al, 6 selfs from A2, 46 hybrids from the union of Al oogonium with A2 antheridium, and 92 hybrids resulting from the union of A2 oogonium with Al antheridium.
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    Characterization of a new virus isolated from pineapple
    ( 1989) Gunasinghe, Ukkubandage
    Mealybug-wilt of pineapple is one of the serious diseases in pineapple. Severe mealybug infestation may result in large yield losses in commercial plantations. The cause of this disease has been linked to a toxin secreted by mealybugs during their feeding upon pineapple. The biological evidence supporting a viral etiology began to emerge in recent years. In this study I investigated the possible involvement of a virus with this disease by studying the association of dsRNA with infected plants, developing purification protocols for the closterovirus-like particles associated with mealybug-wilt infected pineapple, conducting detailed electron microscopic studie6 of the virus leading to the determination of its physical properties such as length, width, and buoyant density. A polyclonal antiserum was produced to the virus isolated from pineapple and agar diffusion tests and serologically specific electron microscopic studies were conducted on this virus. The viral coat protein was analyzed by SDS-polyacrylamide gel electrophoresis. That the antiserum produced was specific to the viral coat protein was shown by western blotting studies. Complementary DNA (cDNA) probes were developed and were used for the detection of virus in pineapple plants, mealybugs, and other plant species found commonly near commercial pineapple plantations. cDNA probes also were used in Northern blot analysis to detect subgenomic RNAs of viral origin in plant extracts obtained from diseased pineapple plants. cDNA probes developed were sensitive and virus was detected in wilt-affected pineapple plants as well as symptomless pineapple plants in the commercial plantations. The presence of virus also was detected in mealybugs taken from diseased pineapple plants and in a common grass species around pineapple plantations. The results of this study confirm the presence of a virus associated with "mealybug-wilted" pineapple plants. Based on the virion morphology and its physical properties this virus may tentatively be assigned to the closterovirus group. The role of the virus in the etiology of wilt disease has yet to be determined.
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    Oosporogenesis and chlamydospore formation in Phytophthora capsici
    ( 1984) Uchida, Janice Y.
    The sexuality of Phytophthora capsici was studied in 30 isolates. with cultures from solanaceous and nonsolanaceous hosts. including Leonian's type culture. Of the 30 isolates. 4 belonged to the Al compatibility type, 7 were A2 and 19 were infertile or AO. Fertility varied among the 11 fertile isolates from highly fertile to very low in fertility. Seven of the 11 isolates produced a few oospores in unpaired (solo) cultures. Although P. capsici has been considered a heterothallic species. oospore distribution patterns in pairings between compatible Al and A2 isolates suggested mutual induction of oospore formation. Oogonia and oospores which formed near the A2 colony were smaller than those developing near the Al colony suggesting that the smaller bodies were A2 products and the Jarger bodies were formed by the Al isolate. This was confirmed in pairings physically separated by polycarbonate membranes. Several oosporogenic pathways in P. capsici were distinguished and five categories were suggested for the genus: (1) homothallism sensu stricto. (2) induced homothallism. (3) noninduced homothallism; (4) secondary homothallism and (5) heterothallism. The optimal temperature for oospore formation in darkness was 20-24 C. whereas 31 C was distinctly inhibitory to oospore production. Light inhibition of oospore formation was found to be temperature dependent. Effect of light over the spectrum from 290 to 750 nm was minimal at 16 C, while at 24 C there was pronounced inhibition at 440-470 nm with some inhibition between 500 and 700 nm. Indirect effects of light were discovered in comparisons of several culture media used to study oosporogenesis. Clarified vegetable juice agar, oatmeal agar, and a synthetic medium, inhibited oospore formation when these were exposed to light prior to inoculation with the Al and A2 compatibility types. Rape-malt agar was not affected by light under the present test conditions. Phytophthora capsici produced abundant oospores when Difco Bacto agar was used as the solidifying agent but oospore production was greatly reduced on Seakem HGT(P) agarose, Difco Noble agar or Difco Purified agar. Chlamydospores were found in 20 of 29 isolates of P. capsici using a submerged culture method. Chlamydospores were not produced in the papaya fruit broth method developed for P. palmivora. The ready production of chlamydospores in most of these isolates of P. capsici suggested a need to expand the original description to provide for the presence of chlamydospores in some isolates.
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    Factors influencing the population dynamics of Meloidogyne konaensis on coffee in Hawaii
    (University of Hawaii at Manoa, 2003-05) Serracin, Mario ; Schmitt, Donald P ; Botanical Sciences (Plant Pathology)
    Experiments were conducted in the greenhouse, field and growth chambers to evaluate effects of soil type, soil moisture regimes, and porosity on selected aspects of the dynamics of the Kona coffee root-knot nematode, Meloidogyne konaensis. First, the reproduction and damage potential of M. konaensis on resistant and susceptible rootstocks of coffee in four soils under two moisture regimes representative of areas where coffee is grown in Hawaii were assessed in greenhouse experiments. M. konaensis suppressed growth of coffee in all four soils. Nematode reproduction occurred readily in all soil types. Reproduction was lowest in the Hydric Dystrandept soil where the nematode holotype was found. In contrast, root galling was greatest in this soil. Greater galling occurred under constant moisture (33kPa) than under fluctuating moisture conditions in this soil. A field experiment in Kainaliu, Hawaii was conducted to determine the influence of irrigation, plant age, cultivar and nematode on coffee growth and yield. The population densities of the nematode in the soil varied according to plant age and irrigation treatment. Soil populations under irrigated conditions were greater during the months of May to July which normally follows the greatest annual precipitation and a period of active plant growth. Nematode reproduction was greater on coffee transplanted as 6-month-old seedlings than on coffee transplanted at 12- month ofage. Soil water tension varied by season and experimental treatment. Trees from 12-month-old transplants exhibited greater water tension fluctuation with greatest water tension occurring from January to April. Trees transplanted as 6-month-old seedlings into M. konaensis infested soil and irrigated yielded greater coffee fruit than the same aged trees treatment without irrigation. Crop loss and reduction of growth and yield were also more evident from 6-month-old seedlings without supplemental irrigation treatment. In contrast, yield from plots in treatments including irrigation, nematode and 12-month-old transplants yielded poorly. Overall highest yields were obtained from trees free of nematode and with supplemental irrigation. Yield reductions from nematode-infected plants ranged from 30-60% which is economically significant. Penetration, development and reproduction of M. Iwnaensis was determined on tomato as model plant at 0.77 and 0.65 porosity. The rate of root penetration and post-embryonic developmental rates occurred slightly faster the porosity treatment of 0.77 than in the more densely packed soil (porosity of 0.65). Development in the 0.65 porosity progressed slower than at 0.77. Even though the nematodes matured faster and began laying eggs sooner on plants growing at porosity of 0.77, much greater numbers of eggs were laid by 30 days after inoculation at the 0.65 porosity treatment than those at the 0.77 porosity. The finding from this research illustrates the primary role of the Kona coffee root-knot nematode in the Coffee Decline. The soil environment and host suitability are conducive factors for the coffee decline disease. Proper soil moisture management combined with sources of genetic resistance could minimize the damage enabling the coffee industry to remain profitable.