Abstract: An interspecific cross was attempted between two homothallic species of Phytophthora, P. sojae and P. vignae. From 1640 single-oospore cultures isolated, DNA was extracted from 800, and two interspecific F1 hybrids (F11121 and F11426) were putatively identified using RAPD markers. The true hybrid nature of these F1 hybrids was confirmed using additional AFLP analysis. Single-zoospore cultures were generated for each F1 hybrid and one single-zoospore culture of each was used in pathogenicity and virulence tests. Both F1 hybrids were pathogenic to soybean and cowpea, causing symptoms including lesions, wilting and death of susceptible soybean and cowpea cultivars. However, the aggressiveness of the F1 hybrids was reduced and was substantially more variable when compared with that of the parental isolates on their respective hosts. The F1 hybrids were reisolated from infected seedlings and their hybrid nature confirmed using RAPD and AFLP analysis. These results provide a basis for further research aimed at obtaining an increased understanding of the genetics of host specificity in the Oomycetes.
Abstract: A new race of Phytophthora vignae f. sp. adzukicola, designated race 4, is reported from central and western Hokkaido, Japan. The isolates obtained from diseased plants of a new cultivar, cv. Syumari, which is resistant to races 1, 2, and 3, were determined to be a new race by the pathogenic reaction on a set of differential adzuki bean cultivars (cv. Erimo-shozu, cv. Kotobuki-shozu, cv. Noto-shozu, cv. Urasa-shimane, and cv. Syumari).
Abstract: Until now gametangia have not been obtained between paired European A1 and American A2 isolates of Phytopthora ramorum in vitro. Their production in artificial culture relies on interspecific pairings. Using P. drechsleri and P. cambivora testers, 51 of 110 P. ramorum isolates from across Europe were all shown to be A1s; while 32 of 38 American isolates from across California and southwest Oregon were shown to be A2s. However, these interspecific pairings are complex, unusually slow and unpredictable. A range of culture media and conditions are described that were tested, unsuccessfully, with a view to enhancing the efficiency of the interspecific pairings. In further tests, gametangia were obtained between A1 and A2 isolates of P. ramorum when juvenile, pre-chlamydospore producing mycelia were mixed together on carrot agar. The gametangia formed in 3–10 d, sparsely to frequently, initially only within the boundaries of the mixed inocula but subsequently in the extended mycelial growth. Chlamydospores were also produced. This inoculum-mixing method, though again sometimes unpredictable, should enhance efficiency of testing for compatibility types and facilitate further studies on whether the sexual outcrossing system of P. ramorum is functional. Differences between sexual reproduction of P. ramorum and that of other heterothallic Phytophthora species are discussed.
Abstract: A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.
Abstract: A dilution-plate technique using five media selective for Phytophthora was evaluated at 16 and 26°C to develop a direct quantitative isolation method for Phytophthora palmivora Butler from naturally infested soil. P10 ARP+H medium at 26°C was found to be the most effective. This method was used to examine the relationship between inoculum density of P. palmivora and disease in papaw seedlings in the glasshouse. Results showed 100% plant mortality at an initial inoculum level of 100.4 cfu g-1 and significant primary root damage (P < 0.05) at ≥2.9 cfu g-1 after 10 weeks in naturally infested soil. Low to medium initial inoculum levels increased during the experiment by four to six times and the highest initial inoculum level increased by two-fold. A survey of 35 papaw-growing sites showed populations of P. palmivora were highest where growers followed papaw with papaw. In most cases, lengthy rotations with other crops and fallows reduced both inoculum levels and the incidence of tree lodging due to root rot.
Abstract: Two methods of isolation, direct plating on selective agar medium and baiting with cocoa pod husks, were used to isolate Phytophthora megakarya from root pieces of some shade trees. Isolates were identified on the basis of their growth rates, colony morphology and sporangium characteristics. Pathogenicity tests were conducted on detached green mature cocoa pods and stems of the relevant host trees. After 36 months of sampling and baiting, P. megakarya was isolated from the roots of four out of 34 shade tree species examined. The host trees were Funtumia elastica (Apocynaceae), Sterculia tragacantha (Sterculiaceae), Dracaena mannii (Agavaceae) and Ricinodendron heudelotii (Euphorbiaceae). P. megakarya isolations were made in both the dry and wet seasons. The rate of recoveries were very low in both seasons ranging from 0.6% to 1.2%. The highest recoveries were in October and the lowest in December and February. In general, plating onto medium was slightly superior to cocoa pod husk baiting for the recovery of P. megakarya. Colonies of P. megakarya isolates from the trees were morphologically indistinguishable from a reference isolate, but were less virulent on cocoa pods than the reference isolate from cocoa. The epidemiological significance of these findings are not clear, but roots of the host trees were likely to be sites for survival and not for multiplication of P. megakarya. Field observation indicated that levels of black pod incidence on cocoa trees around the affected shade trees were not greater than those in other parts of cocoa plantation. This is the first reported isolations of P. megakarya from roots of plants other than cocoa.
Abstract: Indonesia is one of the world’s leading producers of vanilla, an important cash crop for smallholders. Stem rot disease is a major constraint to vanilla production in Indonesia and has caused significant economic losses over the last decade. Previous reports of vanilla stem rots in the Asia-Pacific region include those caused by Fusarium, Colletotrichum and Phytophthora species. In this paper, we report Fusarium species associated with the disease. Seven major vanilla-producing provinces were surveyed for disease incidence and 850 samples were collected. Isolates were recovered from diseased stem tissues using a selective medium. Pure cultures on carnation leaf-piece agar and potato dextrose agar were identified based on morphological criteria. Some ambiguous species were verified based on DNA sequences of the translation elongation factor gene. A total of 542 Fusarium isolates were recovered, comprising 12 species, namely F. decemcellulare, F. fujikuroi, F. graminearum, F. mangiferae, F. napiforme, F. oxysporum, F. polyphialidicum, F. proliferatum, F. pseudocircinatum, F. semitectum, F. solani and F. subglutinans. F. oxysporum was the most commonly isolated species from all areas surveyed, followed by F. solani and F. semitectum. F. oxysporum, F. solani and F. semitectum were tested for pathogenicity to vanilla but only F. oxysporum was shown to be pathogenic. The vanilla stem rot pathogen in Indonesia is verified to be F. oxysporum f. sp. vanillae.
Abstract: Infection by Phytophthora ramorum was associated with stem and leaf lesions of Pacific madrone (Arbutus menziesii) seedlings and saplings. In addition, a common and native pathogen, Botryosphaeria dothidea, caused similar leaf and stem lesions. When exposed to natural levels
of inoculum in forests infested with P. ramorum, 50 to 66% of madrone saplings used as bait died. Recovery of P. ramorum from colonized plant tissue on culture media was generally low. From initial infection, P. ramorum was not culturable from leaf tissue after a mean of 3.5 weeks
or from stem tissue after a mean of 8 weeks. Generally, B. dothidea was recovered more frequently from necrotic stems and leaves than was P. ramorum. Experimental inoculations of madrone seedlings showed that leaf and stem lesion lengths were, on average, greater on tree seedlings inoculated with P. ramorum than on those inoculated with B. dothidea. P. ramorum and <>B. dothidea appear to coexist in stem and leaf tissue, forming a novel pathogen complex, affecting growth and reproduction of Pacific madrone.
Abstract: Appressorium formation is believed to be an important event in establishing a successful interaction between the late blight pathogen, Phytophthora infestans, and its host plants potato and tomato. An understanding of molecular events occurring in appressorium development could suggest new strategies for controlling late blight. We used parallel studies of the transcriptome and proteome to identify genes and proteins that are up-regulated in germinating cysts developing appressoria. As a result, five distinct genes involved in amino acid biosynthesis were identified that show increased expression in germinating cysts with appressoria. These are a methionine synthase (Pi-met1), a ketol-acid reductoisomerase (Pi-kari1), a tryptophan synthase (Pi-trp1), an acetolactate synthase (Pi-als1), and a threonine synthase (Pi-ts1). Four of these P. infestans genes were also up-regulated, although to lower levels, during the early, biotrophic phase of the interaction in potato and all five were considerably up-regulated during the transition (48 hpi) to the necrotrophic phase of the interaction. Real-time RT-PCR revealed that expression of potato homologues of the amino acid biosynthesis genes increased during biotrophic and necrotrophic infection phases. Furthermore, we investigated levels of free amino acids in the pre-infection stages and found that in most cases there was a decrease in free amino acids in zoospores and germinating cysts, relative to sporangia, followed by a sharp increase in germinating cysts with appressoria. Amino acid biosynthesis would appear to be important for pathogenicity in P. infestans, providing a potential metabolic target for chemical control.
Abstract: Genetic study was carried out among 76 isolates of Phytophthora citricola obtained from trees and shrubs belonging to 12 different plant genera grown in more than 10 nurseries. DNA was extracted from pure pathogen cultures and amplified by the PCR technique using ISSR and RAPD primers. The isolates clustered in two main groups. In one of them two subgroups were present. One of those subgroups contained isolates mostly from the family Ericaceae and the second mostly from coniferous plants. Close similarity between isolates from the same nursery was not a rule.
Abstract