Phenotypic differences among single-oospore cultures of Phytophthora palmivora and P. botryosa from Heavea brasiliensis

Oospores ofPhytophthora palmivora andP. botryosa fromHevea brasiliensis were produced when complementary strains of the same species were incubated on V-8 juice agar in continuous darkness, with or without a subsequent period of continuous light. The oospores germinated at a rate of 15–30 % in demineralised water at 26 °C in normal daylight conditions. Other substrates did not improve the germination rate. Single-zoospore colonies derived from sporangia formed by a single oospore were similar to each other in morphology and in pathogenicity toHevea leaves. Over 400 single-oospore isolates from four intraspecific matings ofP. palmivora, and 102 from one pairing ofP. botryosa, were examined. The progeny differed in morphological appearance, mating behaviour, temperature-growth relations, pathogenicity toHevea leaf petioles and cacao pods, rate of production, shape and size of sporangia and in the abundance of chlamydospores. The progeny from an intraspecific cross ofP. botryosa was more variable, with a few isolates being similar in appearance toP. palmivora, having permanently lost their parental characteristic of producing small oval sporangia in clumps. One isolate in particular was indistinguishable fromP. palmivora in morphology and in its ability to produce functional oospores when mated withP. palmivora. Oospores formed by interspecific crosses could not be germinated. With both species, many progeny was less pathogenic than the parents, and many completely non-infective isolates with respect toHevea, cacao and other host plants were produced. Sexual reproduction resulted in a diversity of phenotypes, and both parental types and recombinants were recovered.     

The colonisation of fallen apple leaves by Phytophthora syringae in relation to inoculum levels in orchard soil

Freshly abscissed apple leaves were rapidly infected after inoculation with zoospores of Phytophthora syringae. Mesophyll was extensively colonised within 4 days, sporangia and sex organs were formed within 9 days, but oospore maturation took several weeks. Oospores developed abundantly in leaves of all nine apple cvs tested. The ability of P. syringae to colonise leaf tissue diminished rapidly as leaves senesced, was prevented if leaves were first killed by freezing or desiccation, but was unaffected by watersoaking leaves before inoculation. Of nine soil samples from five apple orchards analysed for P. syringae by dilution and baiting, seven contained from 7–16 infective units/g, one contained significantly more at 34 units/g and one was even more infectious but unquantifiable. These differences of inoculum content of soils, which were only partially corroborated by successive baitings of bulk soil through leaf fall, are discussed in relation to the timing of leaf fall, the activity of P. syringae and rainfall.     

STRUCTURE AND GERMINATION OF BLASTOCLADIELLA EMERSONII RESISTANT SPORANGIA

Resistant sporangia of Blastocladiella emersonii were induced by the addition of bicarbonate, potassium chloride, sodium chloride, or ammonium chloride to the medium and by the exposure of the zoospores to ultraviolet irradiation. Mature resistant sporangia induced by all of these conditions exhibit similar areolate wall pitting. Under suitable conditions resistant sporangia in all cases examined germinated with the cracking of the outer sporangial wall, with the formation of exit tubes by the inner sporangial wall, and with the cleavage and release of zoospores through discharge papillae formed in the tips of the exit tubes.     

Homothallic sexual reproduction of Pustula helianthicola and germination of oospores

Sunflower white blister rust has become an important disease in many countries with intensive cultivation of the important oil crop. The biology of the pathogen is still partly unclear, particular with respect to its sexual reproduction and primary mode of infection. Zoospores released from sporangia of Pustula helianthicola were isolated individually and used for the inoculation of sunflower in order to generate unithallic, genetically homogenous infections. Single zoospore inoculation of young seedlings resulted in mitotic sporulation within subepidermal blisters on cotyledons and true leaves after approximately 2 weeks. Three weeks postinoculation, the infected plants started forming oospores, hence indicating homothallic sexual reproduction of the pathogen. The development of oogonia and antheridia was studied using light and fluorescence microscopy. Oospores were isolated from infected plant tissue and used for infection and germination studies. Microscopic observation of isolated oospores showed germination that formed sessile vesicle-like structures, germ sporangia or only germ tubes. The rate of germination reached approximately 40 %. Germination was not dependant on a resting phase after oospore formation. Oospores applied to the above ground parts of sunflower seedlings lead to infections within a similar time frame as was achieved with mitotic sporangia. The results underline the importance of oospores for primary infection at the beginning of the season and for long-distance dispersal of the pathogen with sunflower seeds contaminated by oospores.     

MORPHOLOGICAL AND ECOLOGICAL STUDY OF PHYSODERMA DULICHII

A new species of aquatic Phycomycete, Physoderma dulichii Johns, parasitic on the aquatic sedge Dulichium arundinaceum (L.) Britt., is described from northern Michigan. This parasite infects and kills the upper epidermal cells of the host leaves. Macroscopically, infection by P. dulichii is indicated by striking brown bands with irregular margins, at intervals on the upper surfaces of the leaves. Like other species of Physoderma, this organism's development includes two distinct phases, an epibiotic monocentric phase producing asexual zoospores and an endobiotic polycentric phase bearing thick-walled resting spores that germinate after an extensive period of maturation at low temperature to form zoospores. The morphology and development of the two phases and of resting spore germination are reported in detail. Only the immature leaves of the host are susceptible to infection, which may be initiated by the introduction of mature resting spores, zoospores from germinated resting spores, or zoospores from epibiotic sporangia. Resting-spore zoospores may also produce the endobiotic stage directly. Initiation of infection in nature requires that the terminal cluster of immature leaves on the host plant be submerged, but infection of subsequently formed leaves of emergent culms can be accomplished through the agency of zoospores from epibiotic sporangia on older leaves. The relation of infected stands of hosts to their environment is discussed and the importance of standing water to infection noted. The geographical distribution of the parasite shows correlation with the drainage basins of the Great Lakes, the St. Lawrence River, and the northern Atlantic Coastal Plain     

ANNUAL GERMINATION WINFOW IN OOSPORES OF NITELLA FURCATA (CHAROPHYCEAE)1

Oospores of Nitella furcata subsp. megacarpa (Allen emend. Wood) were collected from an oospore bank in the sediments of Lake George, New York. Incubated at constant temperatures, all or nearly all of the oospores germinated when exposed to a brief pulse of red light when the annual window of germinability was open. The window seems related to the annual cycle of sediment temperatures. It is open in spring and closes wit the onset of a secondary dormacncy in the summer. Oospores in storage follow a parallel path if held at 18°C, a summer equivalent temperature; the window remains open indefinitely if the oospores are held at 4°C. Attention is drawn to the similarity if the cyclic window of germinability in seeds of summer annuals and oospores of N. furcata.     

Agarose Medium for Germination of Oospores of Phytophthora cactorum

When oospores of Phytophthora caetorum from 30-day-old culture were treated with 0.25% KMnO₄ for 20 min and incubated at 24°C under light for 10 days, 65–75% germinated on water agar and water agarose but only 1–21% germinated on V-8 agar and S+L agar. Water agarose was selected because germinated oospores formed restrieted colonies on this medium that could be isolated easily. KMnO₄ treatment killed sporangia, chlamydospores and mycelial fragments present in oospore suspensions. Under the above conditions, approximately 44% of oospores from 10-day-old culture germinated and the optimum germination rate of about 75% was obtained when oospores reached about 20 days old.     

A spectrophotometric approach for determining sporangium and zoospore viability of Plasmopara viticola

A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2- to 3-min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore-releasing pathogens.     

Location of Oospores in Buckwheat Seed and Probable Roles of Oospores and Conidia of Peronospora ducometi in the Disease Cycle on Buchwheat

Oospores of Peronospora ducometi, the causal agent of downy mildew of buckwheat (Fagopyrum esculentum), were found in the calyx remnant attached to the seed, on the inside of the seedcoat and in the spermoderm layer between the seedcoat and the endosperm. This constitutes a first report documenting the location of oospores in buckwheat seed. Systemic infection of seedlings occurred from oospore-infested seed. Conidial germination was greater at 14°C than 25°C. Systemic infection also occurred as the result of conidial infection of leaves. It is proposed that primary infection of buckwheat occurs by the germination of seed-borne oospores resulting in systemic invasion of the seedling by the germtubes, and followed by conidial formation on the cotyledons. Secondary infection occurs initially from conidia produced on the cotyledons as a result of the systemic infection from seed and subsequently as the result of repeated infections by conidia produced on leaf lesions as the disease progresses up the plant.     

Epidemiology of Cladosporium allii and Cladosporium allii-cepae, leaf blotch pathogens of leek and onion.

Conidia of Cladosporium allii and C. allii-cepae germinated over the temperature range 2–30°C on agar with optimal responses at 15–20°C (C. allii) and 20°C (C. allii-cepae). Conidia of both fungi germinated in water and at c. 100% relative humidity (r.h.) but not at lower humidities on leaf and glass slide surfaces. Germination was more rapid when spores were applied dry to agar or leaves than when applied in water or nutrient solution. More lesions developed when conidia of C. allii-cepae were deposited dry on onion leaf discs or leaf surfaces than when they were applied suspended in water. Conidia of both fungi required 18–20 h at c. 100% r.h. to germinate and infect when applied dry to leaves. Damaging the leaves or the addition of nutrients to the leaf surface increased the incidence of infection by C. allii-cepae compared to controls. Inoculated onion bait plants placed out-of-doors developed infection after at least 17 h at c. 100% r.h. or with leaf wetness. Similar conditions were necessary for infection of bait plants exposed in onion and leek crops infected by C. allii-cepae and C. allii respectively. Disease development and spread of infection occurred at different rates over the same period in two different cultivars of leeks, with spore concentrations increasing in proportion to disease. Spore numbers in the air fell considerably when infected leeks were ploughed under.     

The biology of Peronospora viciae on pea: laboratory experiments on the effects of temperature, relative humidity and light on the production, germination and infectivity of sporangia

Germination of Peronospora viciae sporangia washed off infected leaves varied from 20% to 60%. Sporangia shaken off in the dry state gave 11–19% germination. Most sporangia lost viability within 3 days after being shed, though a few survived at least 5 days. Infected leaves could produce sporangia up to 6 weeks after infection, and sporulating lesions carried viable sporangia for 3 weeks. Sporangia germinated over the range 1–24 °C, with an optimum between 4 and 8 °C. Light and no effct. The temperature limits for infection were the same as for germination, but with an optimum between 12 and 20 °C. A minimum leaf-wetness period of 4h was required, and was independent of temperature over the range 4–24 °C. Maximum infectivity occurred after 6h leaf wetness at temperatures between 8 and 20 °C. Infection occurred equally in continuous light or in darkness. After an incubation period of 6–10 days sporangia were produced on infected leaves at temperatures between 4 and 24 °C, with an optimum of 12–20 °C. Exposure to temperatures of 20–24 °C for 10 days reduced subsequent sporulation. Sporangia produced at suboptimal temperatures were larger, and at 20 °C. smaller, than those produce at 12–16 °C. Viability was also reduced. No sporangia were produced in continuous light, or at relative humidities below 91%. For maximum sporulaiton an r.h. of 100% was required, following a lower r.h. during incubation. Oospores wre commonly formed in sporulating lesions, and also where conditons limited or prevented sporulation. The results are discussed briefly in relaiton to disease development under field conditions.     

In planta selfing and oospore production of Phytophthora cinnamomi in the presence of Acacia pulchella

This paper provides the first evidence of A2 type 1 and type 2 isolates of Phytophthora cinnamomi producing selfed oospores in planta in an Australian soil and in a potting mix. Oospores were observed in infected lupin (Lupinus angustifolius) roots incubated for 7 d either in the substrate under potted Acacia pulchella plants, or in soils collected from under and near varieties of A. pulchella in jarrah (Eucalyptus marginata) forest. The A2 type isolates varied in their ability to produce selfed oospores and none were produced by A1 isolates. The gametangial association was amphigynous and spores were predominantly spherical with diameters from 13–40 μm. The oospores were viable but dormant. Two A2 type isolates produced small numbers of selfed oospores with amphigynous antheridia axenically in Ribeiro's liquid medium within 30 d, and one A2 type 2 isolate produced oospores after mating with an A1 strain. Evidence is presented that the presence of roots of Acacia pulchella, and particularly A. pulchella var. glaberrima and var. goadbyi, enhances the production of oospores.     

Production of Phytophthora infestans oospores in planta and inoculum potential of in vitro produced oospores under temperate highlands and subtropical plains of India

High moisture content of the host tissue ( 88%) and low ambient r.h. (50-54%) favoured oospore formation under controlled environments. It took 14–16 days for oospores to develop; thereafter the number of oospores increased with time and decreased with moisture content of host tissue. High ambient r.h. (> 80%) did not favour oospore formation under field or controlled conditions. Oospore formation was detected in inoculated plants grown in the field when the ambient r.h. declined to 74% and moisture content of host tissue decreased to 83.7–85.6%. It took 8 days (cv. Kufri Chandramukhi) to 13 days (cv. Kufri Jyoti and Kufri Badshah) for oospores to develop. Cultivars also differed in their response to oospore production, cv. Kufri Chandramukhi being more responsive (4800 oospores g⁻¹ f wt) than cv. Kufri Jyoti and Kufri Badshah (1320 and 390 oospores g⁻¹ f wt respectively). Oospores produced in vitro remained viable when buried in soil in the temperate highlands of Himachal Pradesh and sub-tropical plains of Uttar Pradesh, India for more than 150 days, i.e. beginning of the next crop season. The oospores germinated and initiated late blight infection at the base of the stems after 21–30 days of incubation of the potato plants raised in oospore-infested soil. It took 2 days for newly formed oospores to germinate and this delay time increased to 75–77 days after 180-days burial. It took 15 days for their germination (47%) in soil extract as compared to 50 days in sterilised distilled water.     

Survival, Germinability and Infectivity of Oospores of Peronospora viciae f.sp. fabae

A series of experiments was conducted to germinate oospores of Peronospora viciae f.sp. fabae. With rare exceptions, dry-stored oospores did not germinate in water nor did they infect faba bean seedlings in soil. Long-term storage, pre-treatment with KMnO₄ or addition of nutrients to the medium did not induce germination. Survival and infectivity of dry-stored oospores were compared to those of oospores incorporated in a silt loam and a loamy sand soil in the field during 21–22 months. Under dry conditions, the percentage of living oospores did not change as determined by the vital stain tetrazolium bromide. In soil, less than 2% of the oospores had survived after 21 months. Infectivity of oospores was determined by a bioassay 17 and 21 months after oospores had been incorporated in soil. Diseased seedlings were obtained after inoculation of faba bean seeds with oospores extracted from the soil but not with the drystored ones. Soil samples from two field plots naturally infested with oospores 2 and 3 years before the bioassay were infective. Oospores collected with diseased plant material on one of these plots and subsequently stored dry for 3 years were not infective. The results suggested that oospores need a period of natural weathering to become germinable and infective.     

Post-harvest treatments for the control of Phytophthora syringae storage rot of apples

Oospores of Phytophthora syringae germinating at 10 and 15°C under artificial light formed one or more sporangia, which yielded zoospores. Furalaxyl and metalaxyl demonstrated eradicant action against infections occurring on zoospore-inoculated apples. Control was obtained at 10°C when treatment was delayed 5–11 days after inoculation. Storage in 5% CO₂+ 3% O₂ and 0% CO₂+ 2% O₂ reduced rotting compared with storage in air.     

Splash dispersal of fungus spores and fungicides in the laboratory and greenhouse

A laboratory technique is described for the production of drops of simulated rain in which fungal spores were suspended. When such drops containing conidia of Botrytis fabae impacted on a target leaf the secondary droplets produced infections on receptor broad bean leaves. The capacity of fungicides applied to the target leaf to redistribute in secondary splash droplets was examined in terms of the infectivity of the spores in the droplets. The extent to which a copper fungicide reduced infection on the receptor leaves was related to the level and tenacity of the fungicide deposit on the target leaf. The effect of wetting agents on the redistribution of this fungicide could probably be explained by their influence on the tenacity of the initial deposit. In general the capacity of different fungicides to inhibit infection by the secondary droplets was related to the inherent toxicity of the fungicides to B. fabae. Implications of the dispersal of spores and fungicides by rain splash are briefly considered with reference to field conditions.     

The biology of Peronospora viciae on pea: factors affecting the susceptibility of plants to local infection and systemic colonization

Peronospora viciae (Berk.) Casp. penetrated leaf disks of Pisum sativum L. through the cuticle. Resistance of pea plants and of individual leaves to infection by P. viciae increased with age, but decreased again at senescence. Resistance was shown by a restriction in fungal growth and sporulation and by a chlorotic reaction in the leaves. Systemic invasion followed infection of meristematic tissue, and was induced by inoculation into the apical bud of young plants, or on to the epicotyl or hypocotyl, but not roots of germinating seedlings. Most plants whose growth was retarded showed an increased resistance to systemic infection. Pods were infected externally by sporangia, rather than by mycelial growth through the peduncle and pedicel. Oospores and mycelium were found in the testas of some seeds, but seeds from infected pods did not give rise to infected seedlings.     

Susceptibility to purple blotch (Alternaria porri) in garlic (Allium sativum)

Experiments were conducted to study the progress of purple blotch disease of garlic caused by Alternuria porri in the field, to determine the relationship between garlic leaf age and susceptibility to Alternaria porri, and also to assess loss in bulb characters due to purple blotch of garlic. Per cent disease severity and number of purple blotch lesions on four garlic genotypes of known susceptibility, Sel-10 (highly susceptible), G-41 (highly susceptible), IC-49382 (moderately susceptible) and IC-49373 (moderate to less susceptible) were monitored from bulb formation to bulb maturity at weekly intervals. Lesions appeared early on highly susceptible cultivars, Sel-10 and G-41. Rapid progress of disease development was noticed during the last 3 wk before bulb maturity. Peak severity at the maturity of the crop was significantly higher on highly susceptible genotypes. No definite correlation could be established between number of lesions and disease severity. A logistic curve was fitted to predict the disease progress on different weeks before bulb maturity. Levels of leaf tissue found damaged by A. porri at weekly intervals from bulb initiation to bulb maturity were significantly lower on younger leaves than on older leaves. Leaves that emerged 7 wk before bulb maturity required more than a 5 wk period to reach 50% leaf damage, whereas leaves emerging 2, 3 and 4 wk before bulb maturity exceeded 50% leaf damage within a 2–3 wk period. Individual garlic leaves became more susceptible to purple blotch as they aged and emerging leaves were more susceptible the closer they emerged to bulb maturity. Per cent loss in bulb weight and bulb volume was found to be significantly higher on highly susceptible genotypes. No significant reduction in number of cloves/bulb was observed. We propose 4 wk before bulb maturity as the action threshold for initiation of fungicidal application to prevent damaging levels of disease.     

Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro

Pythium myriotylum is the causal organism of Cocoyam Root Rot Disease (CRRD). Significant numbers of zoospores were induced within 1.5 h in cultures in Petri dishes containing P. myriotylum soaked in 0.01 M Ca⁺⁺ and sterile deionized distilled water. Soaking solutions # 2 and # 3 inhibited the production of zoospores of P. myriotylum. This may be due to the delay in maturation of sporangia and the release of zoospores when the soaking solutions contain sucrose. Significant necrosis of detached cocoyam plantlet roots in 100 ml beakers confirmed the infection of zoospores of two `local white' cocoyam genotypes. Detached `yellow' cocoyam roots in 100 ml beakers of genotype RO3015 resisted infection of P. myriotylum with no necrosis of the inoculated roots, which may indicate resistance. This provides a quick and reliable pathogenicity test of P. myriotylum on susceptible cocoyam detached roots. Necrosis of inoculated detached cocoyam roots could be reliably used to screen cocoyam germplasm for resistance to P. myriotylum.     

Stimulation of Photosynthesis in Uninfected Leaves of Rust-infected Leeks

Fourth leaves of healthy leek plants exhibit very low ratesof net photosynthesis which appear to be related to the veryslow growth rates of young leeks early in the season. A stimulation in the rate of net photosynthesis was found tooccur in uninfected leaves of leek plants infected with therust, Puccinia porri. This was accompanied by a reduction inthe rate of photorespiration. Although the mechanisms underlyingthis response are not known, it may be instructive to comparethese results with those obtained from partial defoliation experiments.Increased photosynthetic activity in uninfected leaves may enablethe infected plant to maintain a functional shoot: root equilibrium. Allium porrum L., leek, Puccinia porri, leek rust, stimulation, photosynthesis, reduction, photorespiration