Studies in the biology of Phytophthora cinnamomi Rands

Abstract

1. Sixteen New Zealand and 10 overseas isolates of Phytophthora cinnamomi Rands have been compared for oospore production in paired and single cultures. Considerable variability existed among the 26 isolates in respect of mating ability and responses to physical and chemical factors. Addition of steroids and did not influence oospore production. However, hypoxanthine increased oospore production in two isolates.

2. Discovery of the role of steroids in relation to growth and asexual sporulation of Phytophthora ssp. makes it possible to replace natural media by a synthetic medium. For this purpose an investigation was made into the carbon, nitrogen, vitamin and calcium requirements of P. cinnamomi. Twenty carbon compounds were tested as major sources of carbon with Ca(NO3)2 and glutamic acid as nitrogen sources. Dextrin, starch, and sucrose were the best sources of carbon for growth and asexual reproduction. Xylose also supported abundant sporulation, although growth was slow. Organic acids and alcohols were poor sources of carbon. The best sources of organic nitrogen tested for growth were glutamine and glutamic acid. Tryptophan, phenyl-alanine, and cystine appeared to be inhibitory. Of the vitamins tested, only thiamine was required by the fungus. Five calcium compounds added singly to the basal medium gave no increase in the growth of the fungus.

3. & 4. The effect of soil extracts on sporulation of P. cinnamomi has been investigated. Non-sterile soil extracts from different soil types and from soils under different plan species all supported sporulation, stimulatory activity being markedly reduced only after 1000-fold dilution. Autumn soil was most active in stimulating sporulation, wheras summer soil was inactive. When peptone was added to summer soil, the stimulatory activity of the extract was restored. A wide variety of materials added to distilled water failed to stimulate sporulation. Soil extract lost its stimulatory properties after sterilisation by autoclaving, steaming, Seitz or sintered glass filtration, dialysis, ultrasonic disintegration, ultracentrifugation or treatment with propylene oxide or alcohol. Root exudates and microorganisms other than bacteria had no effect on sporulation. Fifty-nine bacterial cultures classified in 15 genera stimulated sporulation on one or more occasions when transferred from nutrient broth cultures but not from nutrient agar slopes. Of 16 morphologically distinct bacteria isolated from wet autumn soil, two Pseudomonas species stimulated abundant sporulation when transferred from agar plates to sterile soil extract. The activity of these isolates was lost after six weeks in culture, but was partially restored by growing them in nutrient broth. Extracts from sterilised soil inoculated with attenuated cultures of these Pseudomonas species were stimulatory but subcultures from these extracts were only stimulatory if grown in broth. The difference in ability to stimulate sporulation between cultures from broth and from agar was not due to bacterial numbers, flagellation, or fimbriation, but was associated with the additional nutrient transferred with the bacterial inoculum from a broth culture. Streptomycin was the only one of several antibiotics tested which inhibited sporulation. Irrigation of P. cinnamomi mycelium with continuously-produced sterile filtrate from soil extracts produced sporangia in 48 hours.