Conidia In Water Research Articles

Delayed incidence of stem-end rot and enhanced defences in Aureobasidium pullulans-treated avocado (Persea americana Mill.) fruit

The stem-end rot of avocado, caused predominantly by Lasiodiplodia theobromae, can result in heavy postharvest losses. Application of a cell suspension of Aureobasidium pullulans at the stem-end of unripe fruit delayed (P 0.05) affect disease incidence. L. theobromae, when cultured together with A. pullulans, showed significantly (P < 0.05) reduced radial growth of colony towards A. pullulans at 48 and 72 h and shortened aerial mycelium compared to controls. Neither of the organisms overgrew on each other but their colony margins were in close proximity. Presence of A. pullulans significantly (P < 0.001) reduced germination of conidia in water, and the germ tubes were shorter and showed only emergence. Chitinase, β-1,3-glucanase and antifungal activity of the peel of control fruit declined during ripening. However, there was increased chitinase and β-1,3-glucanase activity in A. pullulans-treated fruits or fruits that were inoculated with L. theobromae after treatment with A. pullulans. β-1,3-glucanase activity increased only slightly in fruits that were inoculated with L. theobromae without treatment. Greater preformed antifungal activity was retained in A. pullulans-treated fruits during ripening. Enhanced activity of chitinase and β-1,3-glucanase and greater retention of preformed antifungal activity may have contributed to the delayed stem-end rot incidence in A. pullulans-treated avocados. Application of A. pullulans, 2 days prior to inoculation and retention of stalk at harvest, appears to have allowed better establishment of A. pullulans on the fruit surface.

Production of Ampelomyces quisqualis conidia in submerged fermentation and improvements in the formulation for increased shelf-life

A strain of Ampelomyces quisqualis (ITA3) was found to be more effective against powdery mildew than the commercial one (CNCM I‒807), however its mass production and formulation need to be developed for practical application as biopesticide. Strains of A. quisqualis are considered to be slow growing fungi with a radial growth rate of a few millimetres a week on solid media and their conidia are known to be difficult to produce in industrial fermenters. The current study optimised nutrient composition and growth conditions for high mycelial growth and conidiation of the ITA3 strain on a cost-effective culture medium and finalised a stable and effective dry formulation for the conidia. Plackett–Burman design and response surface methodology were employed for optimisation of new bioprocesses. In the tested culture media, a 23.2-fold increase in biomass and a 2.2 × 108 conidia yield were obtained with the optimised sugar-based medium, with a time reduction of 43% as compared to original sugar-based medium. The kinetic parameter biomass yield (Yx/s) and specific growth rate (μ) values obtained were 3.29 g cells g−1 substrate and 0.56 days−1, respectively. When fermentation was scaled-up in a fermenter, 3.8 × 107 conidia mL−1 were produced within 15 days. Silica gel powder added to undried conidia in water allowed good storage stability and preserved the biocontrol efficacy of the fungus. The high conidia yield and good biocontrol results achieved in this study with formulated ITA3 conidia are economically sustainable for the industrial production of A. quisqualis.

Diurnal periodicity of conidia of aquatic hyphomycetes in water and entrapment on latex-coated slides in two South Indian streams

ABSTRACTAquatic hyphomycete conidial trapping efficiency by the banyan (F. benghalensis L.) latex-coated glass slides was tested diurnally (3 h intervals) in the Western Ghats (Sampaje) and west coast (Konaje) streams in relation to abiotic factors (humidity, air temperature and water temperature). The conidial trapping efficiency of latex-coated slides was compared with plain glass slides and drift conidia in water. Three methods of assessment showed higher species richness, conidial richness and diversity in Sampaje than in Konaje stream. In both streams, species richness, conidial richness and diversity in latex-coated slides were the highest followed by conidia in water and plain slides. Three-way ANOVA revealed significant differences in overall species and conidial richness between the streams, sampling methods and time of sampling (p < 0.001). Multiple comparisons by Holm–Sidak test revealed significant differences in overall species and conidial richness between Sampaje and Konaje (p < 0.001); latex-coated slides and plain slides (p < 0.001); latex-coated slides and water filtration (p < 0.001); plain slides and water filtration (p < 0.001). Total species, total conidia and diversity assessed by the three methods peaked during 12 am–3 am in Sampaje stream, while during 3 am–6 am in Konaje stream. Cooler conditions due to relatively low water temperature favoured higher diversity of aquatic hyphomycetes in Sampaje than in Konaje stream. The three methods employed in the present study were not biased towards scolecoid or stauroid conidia. The top five species in both streams was composed of both types of conidia corroborating earlier annual or biannual studies in Konaje and Sampaje streams. Thus, assessment of population of aquatic hyphomycetes using banyan latex-coated slides will be advantageous over plain slides and drift conidia in streams.

Efficacy of the entomopathogenic fungus Metarhizium brunneum in controlling the tick Rhipicephalus annulatus under field conditions

High infectivity of entomopathogenic fungi to ticks under laboratory conditions has been demonstrated in many studies. However, the few reports on their use under field conditions demonstrate large variations in their success, often with no clear explanation. The present study evaluated the factors affecting the efficacy of the fungus Metarhizium brunneum against the tick Rhipicephalus (Boophilus) annulatus. It demonstrates how environmental conditions and ground cover affect the efficiency of the fungus under field conditions. During the summer, 93% of tick females exposed to fungus-contaminated ground died within 1 week, whereas during the winter, only 62.2% died within 6 weeks. Nevertheless, the hatchability of their eggs was only 6.1% during the summer and 0.0% during winter. Covering the ground with grass, leaves or gravel improved fungal performance. Aside from killing female ticks, the fungus had a substantial effect on tick fecundity. Fungal infection reduced the proportion of female ticks laying full-size egg masses by up to 91%, and reduced egg hatchability by up to 100%. To reduce the negative effect of outdoor factors on fungal activity, its conidia were mixed with different oils (olive, canola, mineral or paraffin at 10% v/v) and evaluated in both laboratory and field tests for efficacy. All tested oils without conidia sprayed on the sand did not influence tick survival or weight of the laid eggs but significantly reduced egghatchability. Conidia in water with canola or mineral oil spread on agarose and incubated for 18h showed 57% and 0% germination, respectively. Comparing, under laboratory conditions, the effects of adding each of the four oils to conidia in water on ticks demonstrated no effect on female mortality or weight of the laid egg mass, but the percentage of hatched eggs was reduced. In outdoor trials, female ticks placed on the ground sprayed with conidia in water yielded an average of 175 larvae per female and there was no hatching of eggs laid by females placed on ground sprayed with conidia in water with canola or mineral oils.

QPCR Assays for the Detection of Cylindrocladium buxicola in Plant, Water, and Air Samples.

Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the β-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3' minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.

First Report of Leaf Spot of Soybean Caused by Aristastoma guttulosum in China.

In fall, 2008, leaf spots were observed during the flowering stage of the Zhong Huang 13 cultivar of soybean in the fields of Anhui Province, China. The leaf spots were irregularly shaped, necrotic, brown-black, and surrounded by yellow halos. Often, on a given leaf, several spots joined one another to form a large blighted area. Finally, those leaves turned yellow followed by defoliation. Damaged leaves showed scattered black spots (i.e., numerous pycnidia) on the lower side. Fresh material was collected from infected plants and a single spore of the putative causal pathogen was isolated on potato dextrose agar (PDA) and incubated at 25°C during a 12-h dark/light cycle. The isolate produced a white fungal colony and black pycnidia after 30 days. The pycnidia are characterized as globose, dark brown-black, and distinctly papillate, with ostiolar setae, and are more or less straight, unbranched, and tapered at the apex. The conidia are clavate, hyaline, mostly with three transverse septa per cell; conidia are either straight or slightly bent, obviously guttulate, and 16 to 29 × 2.5 to 3.5 μm. This pathogen is similar to other Aristastoma guttulosum Sutton (1964), but with the following differences: (a) it has more than 10 versus 4 to 9 setae; (b) conidia are 16 to 29 × 2.5 to 3.5 μm versus 32 to 42 × 3.9 to 4.6 μm as reported for A. guttulosum (1). Conidia of the Chinese isolate were used to inoculate leaves of soybean. Five soybean leaves from potted plants, 1 month old, were sprayed with a suspension of conidia in water. Conidia were harvested from PDA cultures and the suspension was adjusted to 3 × 105 conidia/ml with a hemocytometer. Five leaves were sprayed with sterile distilled water as controls. Inoculated plants were kept in the greenhouse. All five of the inoculated leaves displayed the same symptoms observed in the fields. The symptoms developed initially as brown pinhead spots on the upper side of the leaves, gradually increasing to large brown spots. These spots were irregularly shaped, brown and necrotic in the center and surrounded by a yellow halo. Black pycnidia appeared after 1 week whereas the controls remained asymptomatic. The pathogen was reisolated from the inoculated soybean leaves according to standard Koch's postulates. Primers ITS1 and ITS4 were used in PCR reactions to amplify the internal transcribed spacer region (ITS) (3). Sequencing was performed using the same primers. The ITS sequence (GenBank Accession No. JF825548.1) for this pathogen (587 bp) was submitted to a BLAST search in GenBank. Since the ITS sequence of the genus Aristastoma has never been previously submitted, results did not show high similarity with any extant GenBank sequences. The genus Aristastoma Tehon (1933) was described by Tehon (2). Five of the species in this genus were described by Sutton (1). The number of septate conidium and lack of obvious guttulate within the conidium are the morphological basis to separate these five species. Morphological features of the pathogen from soybean leaves in China were slightly different from those of A. guttulosum. To our knowledge, this is the first report of leaf spot caused by A. guttulosum on soybean in China.

An Outbreak of Leaf Spot Caused by Corynespora cassiicola on Korean Raspberry in Korea.

In September and October 2010, leaf spots were observed on Korean raspberry (Rubus crataegifolius Bunge) plants in farmers' fields in Hapcheon, Gyeongnam Province, South Korea. Disease incidence ranged from 50 to 80% among fields. Circular- to irregular-shaped spots surrounded by yellow halos occurred frequently on the leaves of Korean raspberry plants. Brown spots became dark with wavy borders and ranged from 20 to 300 mm in diameter. Infected leaves became chlorotic, blighted, and eventually died. Fungal hyphae covered the lesions with abundant conidia and conidiophores. Fresh leaf specimens were collected from infected plants and the putative causal pathogen was isolated onto potato dextrose agar (PDA). A total of 30 isolates of the fungus were collected from diseased plants collected in the field. Fungal colonies were gray to brown on PDA. Colonies formed conidia, 38 to 210 × 8 to 20 μm, which were solitary or catenary, obclavate to cylindrical, smooth, straight or curved, and subhyaline to pale brown or brown. Conidiophores, 98 to 840 × 4 to 12 μm, were slightly or conspicuously swollen at apex, single, simple, straight or slightly flexuous, pale to midbrown, smooth, septate, thick, monotretic, and determinate or in tufts. Morphological characteristics of the fungal specimens were similar to descriptions of Corynespora cassiicola (1). A representative isolate of the pathogen was used to inoculate leaves of Korean raspberry plants for pathogenicity testing. Five leaves of a 3-month-old potted plant were sprayed with a suspension of conidia in water. Conidia were harvested from PDA cultures and adjusted to 2 × 104 conidia/ml with a hemocytometer. Five leaves sprayed with sterile distilled water served as controls. Inoculated plants were placed in a humid chamber with 100% relative humidity at 30°C for 24 h and then moved to a greenhouse. Symptoms similar to those observed in the farmers' fields developed on the inoculated leaves within 12 days, whereas the controls remained asymptomatic. The causal fungus was reisolated from the lesions of inoculated plants to satisfy Koch's postulates. To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA region was amplified and sequenced (3). Amplification of the ITS region generated a 559-bp sequence (GenBank Accession No. JQ340026) with 100% similarity to sequences of C. cassiicola in GenBank (Accession No. GU138988) causing leaf spot on cassava (2). Based on the symptoms, morphological characteristics, pathogenicity, and molecular identification, this fungus was identified as C. cassiicola (1). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on Korean raspberry. The recent occurrence of leaf spot on Korean raspberry suggests that C. cassiicola is spreading widely and posing a serious threat to these plants in Korea.

Development of Dactylaria eudermata Drechsler a predaceous fungi in Meloidogyne incognita

Dactylaria eudermata Drechsler is one of the important predaceous fungi occurring widely in different soils. The fungus produces hyphal bails and network compound in which nematodes are entangled. This hyphal nets as trapping structure which may be single dimensional to two or three dimensional and are formed through repeated hyphal anastomosis, which capture nematodes either through the adhesive material present on the surface of hyphal nets or due to physical entanglement. In the experiment it was observed that inflation of hyphal bails takes 30 to 50 minutes and capturing and killing the nematode by a single conidia in water takes 35–55 hours, were as time required for inflation of hyphal bails and real trapping and killing of a nematode in maize meal (MMA: water (1:10) medium) takes one minute and 30–50 hours respectively.

Effects of drying on the survival of conidiosphores of Metarhizium anisopliae var. acridum Driver &amp; Milner

Metarhizium anisopliae var. acridum Driver & Milner (= Metarhizium flavoviride Gams & Rozsypal), isolate CG423, is being developed as a mycoinsecticide against grasshoppers in Brazil. Conidia were harvested and stored in a drying chamber during a study period of 260 d, in a room at ∼25°C. Water content (dry weight) of fresh and dried conidia was 70 and 4%, respectively. Conidia from fresh and dry preparations produced similar in vitro germination patterns and were both infective of the American grasshopper Schistocerca americana Drury. The drying process greatly enhanced the survival of CG423 conidia, as witnessed by nearly 100% germination rates after more than a 100-d desiccation period. Prolonged 135 and 176-d storage in the drying chamber at room temperature decreased viability to 72.5% and 28.5% respectively. In contrast, fresh conidia sealed in plastic bags and stored at ambient conditions for 52 d were inactive (<10% viable). The low germination rate (28.5%) of conidia exposed to 176 d in dry storage was altered by exposure of the dry conidia to high humidity (96% RH). Dry conidia exposed to a 4-h rehydration period had germination rates of ∼50%, whereas 24-h preincubation at high humidity resulted in ∼90% germination rates. However, the germination rates were reduced at 204 (81%), 323 (70%) and 260 (23%) days in storage. In addition, preincubation of the CG423 dry conidia in water produced similar positive effects on germination as compared to germination of fresh conidia. At 24h post-inoculation, dry conidia produced significantly lower germination rates (21%) than rehydrated conidia (germination rates > 91.7%, p < 0.05). However, after 48h on SDAY plates, the germination rate of dry conidia (88.3%) was comparable to the germination of conidia rehydrated in water, water Tween-20, or in high humidity.

Effects of whole-stream nutrient enrichment on the concentration and abundance of aquatic hyphomycete conidia in transport

The concentrations and relative abundances of aquatic hyphomycete conidia in water were followed during a three-year study in two headwater streams at Coweeta Hydrologic Laboratory, North Carolina, using the membrane-filtration technique. After a one-year pretreatment period, one of the streams was enriched continuously with inorganic nutrients (N+P) for two years while the other stream served as the reference. This ecosystem-level nutrient manipulation resulted in concentrations of aquatic hyphomycete conidia in the water of the treated stream that were 4.5–6.9 times higher than the concentrations observed during the pretreatment period and in the reference stream. Nutrient enrichment led to an increase in the number of fungal species detected on each sampling date. Changes in dominance patterns and relative abundances of individual species also were detected after treatment. Nutrient addition stimulates the reproductive activity of aquatic hyphomycetes, their colonization success and fungal-mediated leaf-litter decomposition. Such changes in the activity of the fungal community might affect higher trophic levels in lotic ecosystems.

Effects of Whole-Stream Nutrient Enrichment on the Concentration and Abundance of Aquatic Hyphomycete Conidia in Transport

The concentrations and relative abundances of aquatic hyphomycete conidia in water were followed during a three-year study in two headwater streams at Coweeta Hydrologic Laboratory, North Carolina, using the membrane-filtration technique. After a one-year pretreatment period, one of the streams was enriched continuously with inorganic nutrients (N+P) for two years while the other stream served as the reference. This ecosystem-level nutrient manipulation resulted in concentrations of aquatic hyphomycete conidia in the water of the treated stream that were 4.5–6.9 times higher than the concentrations observed during the pretreatment period and in the reference stream. Nutrient enrichment led to an increase in the number of fungal species detected on each sampling date. Changes in dominance patterns and relative abundances of individual species also were detected after treatment. Nutrient addition stimulates the reproductive activity of aquatic hyphomycetes, their colonization success and fungal-mediated leaf-litter decomposition. Such changes in the activity of the fungal community might affect higher trophic levels in lotic ecosystems.

A novel technique to inoculate conidia of entomopathogenic fungi and its application for investigation of susceptibility of the Japanese pine sawyer, Monochamus alternatus, to Beauveria bassiana

A novel technique to measure the virulence of an entomopathogenic fungus, Beauveria bassiana by exposing tarsi of adults to dry conidia was developed to evaluate effectiveness of nonwoven fabric strip formulation of this fungus for controlling adults of the Japanese pine sawyer, Monochamus alternatus. To regulate inoculum density without suspending conidia in water, conidia were killed by heating at 100°C for 1 h and a step dilution series of conidia was prepared by mixing dead conidia with live conidia at different ratios. The conidial mixtures were attached to tarsi of CO2-anesthetized adults with a fine hairbrush. The 50% lethal doses determined by this method on 14 d were 5.5×106 conidia/individual for aged adults and 1.9×106 conidia/individual for young adults, and on 30 d were 2.8×105 conidia/individual for aged adults and 2.4×104 conidia/individual for young adults. The number of conidia produced on a nonwoven fabric strip was 3.5×108 conidia/cm2, and 8.5×105 conidia/individual were attached to adult beetles which walked on the strip. Based on these results, the validity of a biological control method for M. alternatus to prevent vectoring of the pine wilt disease is discussed.

Relationships of ultraviolet radiation dose and inactivation of pathogen propagules in water and hydroponic nutrient solutions

Conidia of Fusarium oxysporum f. sp. cyclaminis and zoospores of Pythium aphanidermatum were suspended in water and hydroponic nutrient solutions and treated with various doses of UV radiation (253.7 nm) by means of a collimated-beam apparatus and a flow-through apparatus. Germination and colony-forming ability of spores from treated suspensions were estimated on agar media. Colony-forming ability of F. oxysporum f. sp. cyclaminis decreased logarithmically, with increase in applied dose of UV radiation, and in curves that were 3 to 10 times steeper for conidia in water compared to crop nutrient solutions. In tests in the flow-through apparatus, applied doses required to inactivate 99.90% of conidia in water, in fresh nutrient solution, and in nutrient solutions from pepper and tomato crops were 20, 93, 104, and 254 mW·s·cm−2, respectively. In the collimated-beam apparatus, applied doses to inactivate 99.90% of conidia were two to three times higher. Colony-forming ability of P. aphanidermatum zoospores decreased logarithmically, with UV radiation dose, in curves that ranged from similar to twice as steep for zoospores in water compared to plant nutrient solutions. Inactivation of 99.99% of zoospores in water, fresh plant nutrient solution, and pepper crop nutrient solution, respectively, required doses of 12, 17, and 38 mW·s·cm−2 as estimated in the flowthrough apparatus, and 34, 46, and 213 mW·s·cm−2 as estimated in the collimated-beam apparatus. Colony-producing ability of zoospores of P. aphanidermatum and an unidentified species of Pythium from hydroponic lettuce declined more rapidly than did germination incidence as UV dose was increased. In conclusion, UV radiation doses of 30-40 and 20-30 mW·s·cm−2 inactivated most conidia of F. oxysporum f. sp. cyclaminis and zoospores of P. aphanidermatum, respectively, when applied to suspensions of the spores in water. For spores in plant nutrient solutions, the applied dose must be increased to compensate for absorbance of UV radiation in the solution

Self-germination inhibitors fromColletotrichum fragariae

The conidial germination ofColletotrichum fragariae in water was dependent on population, size germination was inhibited at higher concentrations of conidia in water. The inhibition was considered to be caused by some germination-inhibiting substances exuded from conidia, since the germination did occur after washing the conidia repeatedly. The germination-inhibiting substances were searched for in the acetone extract of potato-sucrose-agar (PSA) cultures of the fungus and five active compounds were isolated. Their structures were characterized spectroscopically. HPLC analysis indicated that one of the isolated compounds among these was exuded not only from mycelia but also from the conidia in water under crowded conditions. We suggest that these compounds control the conidial germination of the fungi.

Effect of Formulation and Application Method on the Efficacy of Aerial and Submerged Conidia ofMetarhizium flavoviridefor Locust and Grasshopper Control

A study was carried out to investigate the relative infectivity of aerial and submerged conidia of Metarhizium flavoviride to Schistocerca gregaria and Zonocerus variegatus. The effect of formulation and application method on initial infectivity and field persistence of these conidia was investigated. Strain IMI 330189 was highly virulent to S. gregaria but showed relatively low virulence to Z. variegatus. Direct contact with conidia from the initial spray application resulted in 100% mortality of S. gregaria for all formulation and application combinations. The mean survival time of infected locusts was significantly shorter for treatments using a knapsack sprayer containing submerged conidia in water plus 10 ml litre−1 ‘Codacide’™ (seven days), than treatments with aerial conidia in oil using ULV techniques (8.9 days) or submerged conidia in modified (water plus adjuvants) ULV (MULV) (nine days) or in water-based (VLV) applications (9·3 days). Both aerial and submerged conidia persisted long enough in the environment to effect significant mortality via secondary pick-up of spray residue from vegeta-tion. Persistence was greatest in the ULV and MULV treatments, where the oil component of the formulations provided greater protection of the conidia from environmental stresses. The consequences of secondary pick-up of conidia from the different treatments on total mortality from a single application were examined using a simple host–pathogen model. This predicted that the ULV treatment would be much more effective than the other treatments under conditions where direct contact with the spray was limited. The results of these investigations are discussed in the context of development of optimum spray strategies for control of locusts and grasshoppers, and other pests, under different environmental conditions.

Ultra‐violet radiation damage to Metarhizium flavoviride conidia and the protection given by vegetable and mineral oils and chemical sunscreens

Summary Metarhizium flavouiride conidia formulated in oil or water were exposed to simulated solar radiation. Radiation below 320 nm killed conidia and caused delays in the germination of survivors; germination was greater after 48 h of incubation than after 24 h. UV exposure of conidia formulated in oil for 2 h reduced germination from 99% to 37.5% after incubation for 48 h. Exposure of conidia in water to UV for 1 h resulted in 4.7% germination after 24 h incubation compared with 36.5% for conidia formulated in oil. The addition of 1% oxybenzone resulted in 81.9% conidial germination after 3 h exposure and 48 h incubation compared with 28.1% in oil without the sunscreen.

Acute ozone exposure predispose Phaseolus vulgaris beans to botrytis cinerea

The impact of ozone in predisposingPhaseolus vulgaris toBotrytis cinerea has been investigated. One day after 8 h exposures to 0, 120, 180 and 270 μg ozone m−3, primary and trifoliate leaves of four bean cultivars were detached and inoculated with conidia suspended in water or in an inorganic phosphate (Pi) solution. Visible ozone injury increased with increasing ozone concentrations in all cultivars. Primary leaves were more sensitive than trifoliate leaves. Conidia suspended in Pi solution caused lesions on healthy leaves, whereas conidia in water did not. Ozone-injured leaves of all cultivars showed lesions byB. cinerea after inoculations in water. The number of lesions was significantly correlated with ozone injury for primary leaves. After Pi inoculations, the number of lesions on the ozone-sensitive cultivars also increased with increasing ozone concentrations. However, the ozone-tolerant cultivar Groffy showed first a decrease in the Pistimulated infection at the lowest ozone dosages. The trifoliate leaves of all cultivars were less predisposed to the fungus than the primary leaves. The results indicate that realistic concentrations of ozone enhance the predisposition of bean leaves toB. cinerea. The rate of enhancement depends on the level of ozone-induced injury which was influenced by cultivar, leaf and ozone concentrations.

The Response of Peronospora Parasitica to a Liver Medium

Peronospora parasitica Pers. ex Fr. is an obligate parasite on crucifers; attempts at culture have failed. In water the conidia produce whip-like germ tubes, which swell slightly only where they touch the dish. The medium (2) used to culture species of Puccinia (rusts) that are parasites on higher plants reduces the length of the P. parasitica germ tubes unless it is a 1/10 dilution. In the diluted medium, germtube length is no greater than in the control. Since Syncephalis nodosa van Tieghem, a mucoraceous parasite on other Mucorales, has been grown on a medium containing cubes of beef liver (1), the development of P. parasitica on this medium was studied. Washed, autoclaved 2-mm cubes of liver were placed in a 9-cm Petri dish and covered with 20 ml of a mixture of 0.01% tryptone (Difco: pancreatic digest of casein), 0.04% K2HPO4 and 2% agar: a 1/10 dilution of the K2HPO4 and tryptone concentrations used by Ellis (1). Ten ,ug/ml streptomycin (Calbiochem) were added to this medium to retard bacterial growth. After the agar had solidified, drops of a suspension of conidia in water were put on the surface of the agar over each piece of liver. The plates were incubated at 18 C. After 4 da the germ tubes in the control without liver had disintegrated but in a 5-mm-diam zone around the liver pieces, 90% of the germ tubes were growing toward the liver. They grew from the surface toward the bottom of the medium and formed large swellings or lobes within the agar. The closer the germinating spore was to the liver, the larger were the lobes (FIGS. 1 and 2, A-B). The length of the lobed germ tubes was no more on the average than those without liver, but the width was many times greater and the wide germ tubes did not disintegrate even though they were kept at 18 C for several wk

Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia

The rodlet layer of Neurospora crassa macroconidia has been purified and chemically characterized. Sheets of rodlets were released from the conidial surface by vigorously shaking conidia in water. Conidia were removed by filtration and low-speed centrifugation, and the rodlets were recovered from the supernatant by high-speed centrifugation. The rodlet pellet comprised 1.9% of the initial dry weight. Chemical analysis was hampered by the insolubility of the rodlets. They were not solubilized by heating in various protein-denaturing buffers and were only partially dissolved by heating in 1 M NaOH at 100 degrees C for 5 min. Nevertheless, they were found to be largely composed of protein (91%, based on total nitrogen). The major amino acids in acid hydrolysates were aspartic acid, glycine, serine, alanine, half-cystine, and valine. Glucosamine was not detected in acid hydrolysates. The sulfur content was 2.5%, and this could be accounted for in half-cystine and methionine. Carbohydrate comprised just over 2%. The phosphorus content was 0.21%, of which less than one-third was accounted for in phospholipid. The total fatty acid content was 1.0%, most of which could be accounted for by the fatty acids of the phospholipids.

Fungal Spore Germination on Natural and Sterile Soil

SUMMARY: The germination process of spores of several fungi which require exogenous energy-sources was initiated in non-amended natural soil, a medium deficient in energy-yielding substrates. As measured by subsequent germination time on sterilized soil, this phase accounted for about 8 to 25% of the total germination time. In conidia of Penicillium frequentans it was irreversible, was inhibited by temperatures of 1°C and was dependent on water alone. Continued progress towards germ-tube formation required exogenous energy-yielding nutrients. When incubation on sterilized soil was interrupted by exposure to non-amended natural soil or a model system designed to imitate the microbial energy-source sink of natural soil, progress towards germination ceased in several fungi. Progress already made towards germination was maintained if the exposure to deprived conditions was short (about 3 days or less), but if longer the germination process reverted towards the water-dependent phase. The reversal, in P. frequentans conidia, paralleled the loss of 14C from spores labelled with [14C]glucose. When 14C-labelled conidia were incubated in an artificial nutrient sink, the label lost was about equally divided between 14CO2 and non-gaseous 14C-labelled metabolites. Pretreatment of P. frequentans conidia in water stimulated uptake of [14C]glucose. The results support the view that soil fungistasis in many instances is caused by nutrient deprivation.