Effect of temperature and host genotype on the production of inoculum by Phytophthora fragariae var. fragariae from the roots of infected strawberry plants

A bioassay was used to monitor the release of inoculum in drainage water from strawberry plants inoculated with zoospores of Phytophthora fragariae var. fragariae. The fungus was detected in drainage water from plants that had been held at temperatures between 2 and 20 C. but not from plants held at 26°C. The lag phase before secondary inoculum was first released, the maximum and total amounts of inoculum released, and the length of time over which inoculum was released were all greater at the lower temperature regimes, especially those below 10 C. The results were consistent with observations on the effect of temperature on zoospore production from agar discs and on zoospore motility: more zoospores were produced at lower temperatures and they remained motile for longer. From this it is concluded that the inoculum detected consists mainly of motile zoospores. In most experiments with standardized suspensions c. 10-15 were sufficient to initiate infection of the plants in the bioassay. In general, more inoculum was produced by host genotype/fungal isolate combinations in which there were marked root rot symptoms than in combinations in which the host was resistant.     

Effects of hydrostatic pressure,agitation and CO2 stress on Phytophthora nicotianae zoospore survival

BACKGROUND: Phytophthora nicotianae Breda de Haan is a common pathogen of ornamental plants in recycled irrigation systems. In a previous study, annual vinca (Catharanthus roseus Don) inoculated with zoospore suspensions using a CO₂‐pressurized sprayer had less foliage blight than plants inoculated using a hand sprayer. Here, the impact of hydrostatic pressure, agitation and aeration with CO₂ on the survival of P. nicotianae zoospores was examined. RESULTS: Exposure of zoospores to 840 kPa hydrostatic pressure for 8 min or agitation at a mixing intensity (G) of 6483 s^?1 for 4 min at 22–23 °C did not kill zoospores, but resulted in viable cysts. Motile and forcefully encysted zoospores of P. nicotianae were equally infectious on vinca or lupine (Lupinus polyphylus Lindl.). Bubbling CO₂ into zoospore‐infested water at 110.4 mL (0.2 g) min^?1 for 5 min caused 81% reduction in the number of germinated zoospores. Pressure at 630 kPa (16.3 g CO₂) or 70 kPa (3.85 g CO₂) facilitated CO₂ injection and shortened the zoospore inactivation time to 30 s. When air was bubbled through the suspension, germination was similar to the control. CONCLUSIONS: Exposure to CO₂ killed P. nicotianae zoospores in water. Neither pressure nor agitation had an effect on zoospore viability or infectivity. Based on results of this study, the authors designed a recycling CO₂ water treatment system that is currently under evaluation. Copyright © 2010 Society of Chemical Industry     

A rapid microbioassay for discovery of antagonistic bacteria for Phytophthora parasitica var. nicotianae

A simple, rapid, small-scale microbioassay for infection of tobacco seedlings by Phytophthora parasitica var. nicotianae was developed here. This assay uses tobacco seedlings cultivated in petri dishes for a standardized method for quantitation of initial zoospore inocula and high-throughput screening of antagonistic bacteria. Zoospore inocula between 10(2) to 10(5) spores per petri dish were inoculated on 14-day-old tobacco seedlings for the susceptibility test. The optimum inocula was established to be ten thousand zoospores. One hundred and fifty pure culture bacteria with different pigments, growth rates, and morphologies were isolated from rhizosphere soil of tobacco and screened for protective ability against tobacco black shank. Fifteen bacteria presented high activity against P. parasitica on tobacco seedlings. They were identified by Biolog GEN III MicroPlate and distributed as Bacillus amyloliquefaciens, B. licheniformis, Paenibacillus pabuli, B. atrophaeus, B. subtilis, B. pumilus, and B. endophyticus, respectively. Four antagonists chosen randomly from the 15 bacteria all exhibited the same 100% protective activity in planta as that in the petri dishes. This microassay proved to be a rapid, reproducible, and efficient method for screening of potential biological agents or microorganisms and may be useful for studying mechanisms of infection and control of Phytophthora spp. under hydroponic conditions.     

Effects of temperature,zoospore concentration,infection period,and fruit maturity on <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> infection of figs

White powdery rot in figs caused by Phytophthora palmivora is an important disease resulting in severe fruit rot, but is not currently effectively controlled in Japan due to a lack of understanding of its epidemiology. Therefore, the effects of temperature, zoospore concentration, infection period, and fruit maturity on infection of figs were examined by inoculating the fruit with a suspension of P. palmivora zoospores. The zoospores germinated at temperatures from 5 to 35 °C, with the optimum temperature range being 20–35 °C. Germ tube length in zoospore cysts was greatest at 20–30 °C. The disease developed in green figs at temperatures from 20 to 30 °C. Figs inoculated with as few as 10 zoospores per fruit developed severe symptoms at the optimum temperature (25 °C). The minimum infection period required for infection was 2 h at 20–28 °C. All of the figs developed symptoms within an 8 h infection period at 25 or 28 °C, and with a 6 h infection period at 25 °C. All fruit at different stages of development (immature fruit, yellow fruit, and mature fruit) developed symptoms. These results indicate that P. palmivora is capable of infecting figs over a wide range of temperatures, within a short infection period, at a low concentration of zoospores, and at any stage of development. These data could be used to construct forecasting models and develop effective control systems for white powdery rot.     

大豆疫霉卵孢子微量、快速检测方法的建立

采用套筛富集人工接种于土壤中的大豆疫霉卵孢子。结果表明,当接种量为104、103、102、101、100时,富集百分率分别为45.9%、39.6%、11.8%、1.7%、5.6%。采用玻片压碎法提取微量卵孢子中DNA后,采用PCR技术快速进行检测的结果表明,提取10,5,1个卵孢子中DNA进行PCR扩增成功率分别为83.3%、33.3%、0。提取10个卵孢子中DNA进行PCR扩增时,2个处理出现非特异性PCR产物。内切酶MspI能将特异性PCR产物消化为204bp和124bp两个片段;内切酶RsaI能将特异性PCR产物消化为242bp和86bp两个片段。     

Enkele waarnemingen over versmelten van zoösporen bij Phytophthora infestans (Mont.) de Bary

Summary Zoospores ofPhytophthora infestans were seen to fuse in pairs. Generally within 30 min. after zoospore discharge, fusion started by the formation of a connection between touching zoospores. Gradually the connection became shorter and thicker (fig. 1,2 and 3), and within 30 min. the two zoospores united to form a spherical body (fig. 4). The flagella were thrown off just before the compound zoospore assumed the spherical shape. The compound zoospores germinated by a germ tube within 30 min. after their formation. The analogy withCritopoulos' observations on zoospore fusion inP. capsici is discussed.     

The effects of temperature on zoospores of the crook root fungus Spongospora subterranea f.sp. nasturtii

The effect of temperature on the release, survival, encystment and infectivity of Spongospora subterranea f.sp. nasturtii zoospores released from diseased watercress roots was studied. There was rapid, synchronized release of zoospores at 20°C followed by a steep decline in zoospore numbers. A similar trend occurred at 10° C and 15° C, although fewer zoospores were released and the subsequent decline was more gradual. In contrast, at 5°C very low numbers of zoospores were released over the first 5 days. Subsequently, zoospore release increased rapidly over the following 3 days and zoospore numbers were maintained at relatively high levels for a further 6 days. This duration of high zoospore numbers at 5°C was due to prolonged survival rather than continual release. When a uniform zoospore suspension released at IOC was transferred to 20·C zoospore numbers were found to decline rapidly over 6 days; the decline was less rapid at 15° C and IOC, taking 9 and 15 days, respectively. At 5°C zoospore numbers decreased slowly. Few zoospores encysted on roots at 20° C but increased numbers were found at 15°C and 10°C. There were significantly more diseased roots at 10° C than at 5,15 or 20° C. The lowest number of diseased roots was found at 20° C. The correlation between increased seventy of the disease in the field during the winter months and the effect of temperature on zoospores of S. subterranea f.sp. nasturtii is discussed.     

Etiology of a newly described root rot of guar (Cyamopsis tetragonoloba) in Australia caused by Phytophthora cryptogea

An isolate of Phytophthora obtained from diseased roots and stems of guar ( Cyamopsis tetragonoloba ) growing in waterlogged soil from Dalby. Queensland. was identified as Phytophothora cryptogea ( P. drechsleri ). Sex organs were not observed, Pathogenicity to guar seedlings was established under glasshouse conditions either by using a hypocotyl-wound inoculation technique, or adding motile zoospores to seedlings growing under saturated conditions. This appears to be the first report of this disease. Seven isolates of P. drechsleri from hosts other than guar were shown to be pathogenic to guar following hypocotyls-wound inoculation.
The host range of the guar isolate was studied using both hypocotyl-wound inoculation and zoospores. After hypocotyl inoculation, the following species were found to be susceptible^: cantaloupe. cowpea, cucumber, mungbean, pea. pigeon-pea, safflower. sunflower, tomato and watermelon. Slight symptoms were observed on bean and chickpea. When zoospores were used as the inoculum, only pea. pigeon-pea, sunflower and safflower were highly susceptible. A range of guar cultivars and breeding lines were screened for resistance using zoospore inoculum, and although no genotypes were highly resistant, there were significant differences between disease reactions.     

白兰瓜疫病侵染循环、流行因素及防治研究

德雷疫霉(Phytophthora drechslcri)游动孢子接于白兰瓜(honey dew)叶片上,12小时侵入率达19.3%,96小时孢子囊形成数254.3个/cm~2;德雷疫霉 A_1型与辣椒疫霉(Phytoph-thora capsici)A_2型配对,接于白兰瓜叶上形成大量卵孢子。连续降雨和高温,或高温天气给瓜田灌水是疫病流行的决定因素。70%代森锰锌在低浓度时对德雷疫霉、辣椒疫霉菌丝生长、孢子囊和卵孢子形成均有明显的抑制作用。高畦栽培对烂秧防效达89.2%—92.0%,对烂瓜防效达100%。     

Nonphytotoxic Aluminum-Peat Complexes Suppress Phytophthora parasitica

ABSTRACT Amendment of peat-based potting media with Al(2)(SO(4))(3) suppresses damping-off of Vinca (Catharanthus roseus) caused by Phytophthora parasitica. The species of aluminum (Al) responsible for disease suppression have not been identified. The objective of this study was to determine the effects of amount and pH of Al(2)(SO(4))(3) amendment solutions on survival of P. parasitica. In separate experiments, peat was amended with Al(2)(SO(4))(3) solutions adjusted to pH 4 or 6 at either 0.0158 or 0.0079 g of Al per gram of peat. Amended peat was placed in Büchner funnels maintained at -2.5 kPa matric potential. Peat was infested with P. parasitica by placing zero, two, or five colonized Vinca leaf disks in each funnel, and 15 Vinca seeds were placed in each funnel. After 24 h, the matric potential was brought to 0 kPa to induce zoospore release and returned to -2.5 kPa after 24 h. Pathogen populations and stand counts were assessed after 2-week incubation. Al amendment solutions at both pH 4 and 6 reduced pathogen populations at 0.0158 g of Al per gram of peat. Solutions at pH 4 reduced pathogen populations by more than 90% at both inoculum levels; amendment solutions at pH 6 reduced populations by 95% at the low inoculum level and 65% at the high inoculum level. The prevalence of Al(OH)(2)(+) in peat amended with Al(2)(SO(4))(3) solution at pH 6 suggests that ions other than Al(3+) may be responsible for pathogen suppression. Based on the difference in chemical conditions of Al-amended peat and suppressive mineral soils, the mechanism of Al-mediated suppression of plant pathogens is speculated to be different in the two systems. Peat containing Al-peat complexes was chemically suppressive to P. parasitica and may confer Al-mediated suppression of plant pathogens with a nonphytotoxic form of Al.     

Root rot of lavender caused by Phytophthora nicotianae

Lavender (Lavandula angustifolia) plants with rotted roots and discoloured vascular systems consistently yielded cultures of fungi that were identified as Phytophthora nicotianae van Breda de Haan (= P. parasitica). Inoculation experiments using either zoospore suspensions or mycelial fragments were successful in reproducing symptoms originally observed on wilting and dying plants. Lavender is a new host for P. nicotianae.     

Differential Sensitivity of Phytophthora parasitica var. nicotianae and Thielaviopsis basicola to Monomeric Aluminum Species

ABSTRACT Aluminum (Al) is toxic to many plant pathogens, including Thielaviopsis basicola and Phytophthora parasitica var. nicotianae. Because fungi-toxicity of Al has been described in soils over a wide pH range, multiple species of Al may be responsible for pathogen suppression. The goals of this work were to determine the sensitivity of T. basicola and P. para-sitica var. nicotianae to Al over a range of pH values, quantify the toxicity of monomeric Al species to production of sporangia of P. parasitica var. nicotianae and chlamydospores of T. basicola, and detect the accumulation of Al in pathogen structures. A complete factorial treatment design was used with Al levels ranging from 0 to 100 muM and pH levels ranging from 4 to 6 in a minimal salts medium. The chemistry of test solutions was modeled using GEOCHEM-PC. Colonies were grown in 5% carrot broth, and after 1 or 2 days, the nutrient solution was removed, colonies were rinsed with water, and Al test solutions were added to each of four replicate plates. After 2 days, propagules were counted and colonies were stained with the Al-specific, fluorescent stain lumogallion. The oomycete P. parasitica var. nicotianae was sensitive to multiple monomeric Al species, whereas sensitivity of T. basicola to Al was pH-dependent, suggesting that only Al(3+) is responsible for suppression of this fungal pathogen. Chlamydospore production by T. basicola was inhibited at pH values <5.0 and Al levels >20 muM, whereas sporangia production by P. parasitica was inhibited at Al levels as low as 2 muM across all pH values tested. The lumogallion stain was an effective technique for detection of Al in fungal tissues. Aluminum accumulated in sporangia and zoospores of P. parasitica var. nicotianae and in nonmelanized chlamy-dospores of T. basicola, but not in cell walls of either organism. The differential sensitivity of the two organisms may indicate that true fungi respond differently to Al than members of the oomycota, which are more closely related to plants.     

Germinate to exterminate: chemical stimulation of Spongospora subterranea resting spore germination and its potential to diminish soil inoculum

Hoagland's solution (HS), a defined nutrient supplement for plants, has been previously reported to stimulate zoospore release from resting spores of the potato pathogen Spongospora subterranea f. sp. subterranea. This study obtained direct empirical evidence for an increase in zoospore release with HS treatment, and identified Fe‐EDTA as the stimulant component of HS. Stimulation of resting spores by HS and Fe‐EDTA resulted in greater and earlier zoospore release compared to a distilled water control, and in the presence of a susceptible tomato host plant resulted in enhanced root infection. Given the labile nature of S. subterranea zoospores, it was postulated that stimulation of premature release of zoospores from the dormant resting spores in absence of susceptible hosts could reduce soil inoculum levels. In two glasshouse trials in the absence of host plants, both Fe‐EDTA and HS soil treatments reduced S. subterranea soil inoculum levels, providing proof of concept for the ‘germinate to exterminate’ approach to inoculum management.     

A Rapid Microbioassay for Discovery of Novel Fungicides for Phytophthora spp

ABSTRACT A microbioassay was developed for the discovery of compounds that inhibit Phytophthora spp. This assay uses a 96-well format for high-throughput capability and a standardized method for quantitation of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 x 10(5) zoospores per ml using percent transmittance (620 nm). Subsequent growth of mycelia was monitored by measuring optical density (620 nm) at 24-h intervals for 96 h. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth [RPMI], and mineral salts medium) and four zoospore concentrations (10, 100, 1,000, and 10,000 zoospores per ml) were evaluated. Both full- and half-strength RPMI were identified as suitable synthetic media for growing P. nicotianae, and 1,000 zoospores per ml was established as the optimum initial concentration. The assay was used to determine effective concentration values for 50% growth reduction (EC(50)) for seven commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, etridiazole, metalaxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These EC(50) values were compared with those obtained by measuring linear growth of mycelia on fungicide-amended medium. The microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of compounds that inhibit spore germination in P. nicotianae and should be effective for other species of Phytophthora as well. The assay requires relatively small amounts of a test compound and is suitable for the evaluation of natural product samples.     

Repetitive Applications of the Biocontrol Agent Pseudomonas putida 06909-rif/nal and Effects on Populations of Phytophthora parasitica in Citrus Orchards

ABSTRACT Pseudomonas putida 06909-rif/nal was applied repetitively during the irrigation season in two citrus orchards over 3 years. In a mature (50-yearold) commercial citrus orchard covering 2.02 ha, weekly applications of Pseudomonas putida 06909-rif/nal with an in-field fermentor resulted in soil populations that fluctuated between 2.83 log CFU + 1 per g of soil and 4.35 log CFU + 1 per g of soil. Resulting rhizosphere populations of Phytophthora parasitica were significantly reduced in 1999 but not 1997 or 1998. In a newly planted citrus orchard, yearly applications of Pseudomonas putida 06909-rif/nal at the beginning of the irrigation season resulted in high soil populations of Pseudomonas putida 06909-rif/nal that declined rapidly and never reduced the rhizosphere populations of Phytophthora parasitica. When Pseudomonas putida 06909-rif/nal was applied weekly, soil populations increased throughout the 1997 and 1998 irrigation seasons, reaching a maximum in 1998 and remained high throughout the 1999 irrigation season. Rhizosphere populations of Phytophthora parasitica were significantly reduced in 1998. Yearly applications of the fungicide metalaxyl and the nematicide phenamiphos reduced rhizosphere populations of Phytophthora parasitica in 1997 but not in 1998 or 1999. Pseudomonas putida 06909-rif/nal was uniformly distributed throughout the soil profile to a depth of 75 cm in both yearly and weekly applications. When applied through low-volume minisprinklers, Pseudomonas putida 06909-rif/nal was found in aerosols up to 3 m away.     

恶疫霉有性杂交后代的生物学研究

 将2个带有不同抗药性标记的恶疫霉菌株配对,培养36 d后诱导其卵孢子萌发,从3760个单卵孢株中,获得50株带有双亲标记的杂交个体。对其中32株杂交个体的生长速率、有性和无性繁殖能力、卵孢子和游动孢子萌发率、致病力及对高温的耐受力等生物学性状进行测定。结果显示,测试的32株杂交个体在LBA培养基上均能较好地生长,有24株的杂交个体的生长速率介于2亲本之间;在LBA和SL培养基上,大多数杂交个体均能产生较大数量的卵孢子,有37%的杂交个体在SL培养基上产卵孢子能力显著大于双亲,仅有1个杂交个体不产生卵孢子;在人工培养条件下,大多数供试杂交个体可以产生较大数量的孢子囊,其中15株杂交个体产孢子囊能力处于双亲之间;被测的杂交个体产生的卵孢子或游动孢子均可以萌发形成有效的单孢株,有22株的杂交个体的卵孢子萌发率大于50%;被测的杂交个体接种在苹果上都有较强的致病力,有16株杂交个体的致病力显著大于双亲。表明在群体水平上恶疫霉有性杂交后代具有较强的生活能力,提示同宗配合恶疫霉不同菌株间的有性重组,对该种的群体遗传多样性可能具重要作用。     

Effect of high-power monochromatic (pulsed UV laser) and low-power broadband UV radiation on Phytophthora spp. in irrigation water

Cysts or zoospores of Phytophthora capsici, P. citrophthora and P. nicotianae were suspended in distilled water or in recycled irrigation water collected from commercial nurseries. Propagules, suspended in shallow layers of still water in 60 mm-diameter plastic Petri dishes with lids off, were exposed to various UV doses emitted from either an excimer laser or a mercury vapor lamp. The laser emitted a monochromatic (248 nm, 5 eV/pulse), pulsed UV beam with an intensity of 1 to 2 mJ/cm²/pulse with 20-ns pulse durations and a high peak power of 50 to 100 kW/cm²/pulse. The UV lamp was from a commercial water purifier that emitted broadband (continuous) UV radiation at an intensity of ～8 mW/cm². Survival was assessed by culturing aliquots of the treated or non-treated suspensions onto corn meal agar amended with ampicillin (250 ppm) and rifampin (10 ppm), and then counting numbers of developing colonies. The UV dose (as energy per unit area) required to kill propagules was smaller when they were suspended in distilled water than when suspended in recycled nursery water. The reduced kill effectiveness in recycled water appears to be related to UV-absorbing soluble chemicals. Cysts and zoospores were equally susceptible to UV, although the high-peak power pulsed-UV laser source with ultra-short exposure times appeared to have greater kill effectiveness than the conventional Hg-vapor UV lamp. Phytophthora capsici and P. nicotianae isolates were somewhat less sensitive to UV than isolates of P. citrophthora obtained from various hosts and geographical regions. Furthermore, hyphae of Phytophthora spp. were less susceptible to UV than were cysts or zoospores.     

Effect of fungicides on stages of the life cycle of Phytophthora cinnamomi

An assessment was made of the effects of several fungicides on the formation of zoosporangia, the release of zoospores from zoosporangia, zoospore motility, germ tube formation and mycelial growth, by Phytophthora cinnamomi Rands. Compounds active against mycelial growth (such as metalaxyl and furalaxyl), as well as those active against zoospores (such as captafol and zineb), could be detected by a lupin (Lupinus angustifolius L.) assay which has been developed. This assay may be useful as a screening procedure to detect new fungicides.     

Effects of Cellulytic Enzymes on Phytophthora cinnamomi

ABSTRACT Two enzyme systems, cellulase (beta-1,4-glucanase) and laminarinase (beta-1,3-glucanase), were added to soil extracts to simulate (in vitro) lytic components found in mulches suppressive to Phytophthora cinnamomi. Concentration ranges of each enzyme were incubated with Phytophthora cinnamomi mycelium, zoospores, zoospores cysts, and zoospore-infected excised roots to evaluate the roles of each enzyme in potential control of avocado root rot disease. Cellulase significantly retarded the development of zoosporangia and chlamydospores when mycelia were incubated in soil extract containing the enzyme at concentrations greater than 10 units/ml. Zoospore production was also reduced by cellulase but not by laminarinase. Laminarinase had little effect on zoosporangia or chlamydospore formation. At high concentrations, laminarinase was consistently more effective at preventing encystment than cellulase. Chlamydospores preformed in root tips were immune to the lytic effects of all treatments except cellulase at 100 units/ml. Zoospores placed in enzyme solutions and plated on a selective medium survived high cellulase concentrations and formed colonies, but there were fewer surviving zoospores when laminarinase was present at greater than 10 units/ml. Low concentrations of cellulase stimulated infection of excised roots, however, low concentrations of laminarinase prevented infection. Cellulase and laminarinase have different effects on the structures of the Phytophthora cinnamomi life history, however, each enzyme may have a role in reduction of inoculum.     

Waarnemingen over het gedrag van zoösporen uit de zoösporangia vanOlpidium brassicae (wor.) dang

Summary The zoosporangial zoospores ofOlpidium brassicae are uniflagellate (fig. 1). Observations with a phase contrast microscope on living zoospores in watermounts showed that biflagellate zoospores resulted from zoospore fusion. Further, specimens were found with more than two flagella (fig. 2 and 3). As at zoospore discharge only uniflagellate zoospores occur, it may be, assumed that zoospores with more than two flagella are also the result of zoospore fusion. As there may be an analogy betweenO. brassicae andO. viciae,Kusano's explanation of the occurrence of compound zoospores with more than two flagella is discussed.