The survival of Mycosphaerella pinodes on pea haulm buried in soil

Where haulm was left on the soil surface the mycelium of Mycosphaerella pinodes went through cycles of active saprophytic growth, followed by inactive phases during the earlier parts of the year. Burial at either 15 or 25 cm brought about a decrease in survival with time. The majority of the mycelium was on the outer portions of the stem pieces, with only a small amount of penetration into the plant remains. Experiments on root surface colonization showed that mycelium of M. pinodes was capable of growing saprophytically on the host roots in competition with the normal soil microflora.     

Leaf Spots Induced by Ascochyta pisi and Mycosphaerella pinodes

The leaf-spot pathogens, Ascochyta pisi and Mycosphaerella pinodes,both cause limited, necrotic lesions in detached pea leafletssuspended above water. When leaflets were floated on water A.pisi lesions were still limited, but those caused by M. pinodesspread rapidly to occupy all the leaflet tissue. Increasing the numbers of spores in inocula decreased numbersof mature lesions caused by A. pisi, but increased numbers ofspreading lesions caused by M. pinodes. Older leaflets weremore susceptible to both pathogens. Studies of penetration and colonization of leaves with the aidof light and electron microscopy showed that cell-wall-degradingenzymes were involved in the formation of A. pisi lesions andin spreading lesions caused by M. pinodes. There was littleevidence of cell-wall degradation in limited M. pinodes lesionsin which penetration of walls by hyphae seemed to be mechanicalin nature. No physical barriers developed in tissues surrounding limitedlesions. Nevertheless, A. pisi was rarely found beyond the necroticarea. This suggested that tissues beyond the lesion had becomeresistant to the parasite. In contrast, M. pinodes often grewoutside the necrotic area, sometimes many days after this hadstopped growing, but when it did so it caused no more necrosisunless leaflets were placed in conditions in which the spreadingtype of lesion could develop.     

Effects of temperature and inoculum concentration on infection of narcissus bulbs by Fusarium oxysporum f.sp. narcissi

Chlamydospores of Fusarium oxysporum germinated, and mycelium grew on agar, at 10 but not 8°C. Numbers of chlamydospores needed to initiate disease suggest that the principal sources of infection are within the stock of bulbs and not the soil.     

Thermal sensitivity of Phytophthora cinnamomi and long-term effectiveness of soil solarisation to control avocado root rot

Root rot caused by the fungus Phytophthora cinnamomi is a major disease of avocados worldwide. Heat sensitivity of a collection of P. cinnamomi isolates was determined by exposing agar discs containing mycelium or mycelium plus chlamydospores at various temperatures for different periods. Long‐term effectiveness of soil solarisation to control Phytophthora root rot was evaluated in two field trials. In the first, soil disinfestation by solarisation was applied in 1990 to a naturally infested plot before planting avocado (Persea americana) and viñatigo (Persea indica) seedlings. In the second trial, established avocado trees were solarised for four consecutive summers (1996–1999). Results for heat sensitivity showed that fungal mycelium was inactivated after 1–2 h at 38°C. However, 1–2 h at 40°C was needed to kill all propagules when chlamydospores were present. Fungal growth inhibition after thermal treatments was related to levels of time and temperature, and detrimental effects occurred as consequence of sublethal thermal doses. Soil solarisation presented long‐term positive effects when applied as a preplanting treatment. Five years after solarisation, disease severity (0–5 scale where 0 = healthy and 5 = dead plant) of avocado and viñatigo planted in solarised soil was 2.03 and 0.71, respectively, compared with 4.65 and 4.84 in controls. Eleven years after solarisation, the percentage of dead plants in solarised soil was 73% for avocado and 43% for viñatigo but 100% in controls. In contrast, an insufficient level of control was observed in established orchards, probably because of the lower temperature reached during solarisation under the shade of tree canopy. In this situation, maximum temperatures at 5‐cm depth were 10–13.7°C lower than under solar‐heated mulch, only exceeding 40°C in 1997.     

Behaviour of Phytophthora citrophthora and P. nicotianae var. in soil, and Differences in Their Tolerance to Antimicrobial Components of Selective Media Used for Isolation of Phytophthora spp.

Phytophthora citrophthora was inhibited to a greater extent than P. nicotianac var. parasitica by chloramphenicol, hymexazol, PCNB and pimaricin at concentrations used in selective media for the isolation of Phytophthora spp. Reduced concentrations of the antimicrobial components of the selective media to tolerant levels for P. citrophthora markedly increased the recovery of the two brown rot pathogens from soil. Mycelium of both Phytophthora spp. survived in air-dried soil for at least 5 months while mycelium of most Phytophthora spp. do not survive in dry soil. In moist soil, P. nicotianae var. parasitica produced hyphal swellings, sporangia and chlamydospores. P. citrophthora produced hyphal swellings and sporangia, but no chlamydospores. No oospores were produced, even in pairing cultures on agar plates with isolates obtained from several locations of citrus groves andfruits by both species. Sporania were obtained in both species in citrus groves on mycelium mats, in the rhizosphere, in infected leaves and fruits buried at soil depths of 5–35 cm. Numbers of propagules declined during the incubation period, but conside, rable numbers survived throughout the experimental period (6 months). Although P. nicotianae var. parasitica produced chlamydospores while P. citrophthora did not, numbers of surviving propagules recovered from soil after 6 months were comparable with both species. The brown rot pathogens survived in soil both as colonizers of detached leaves and fruits and as parasites in living root tissues.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

Pea seed infection by Mycosphaerella pinodes and Ascochyta pisi and its control by seed soaks in thiram and captan suspensions

Apparently normal pea seeds from pods bearing lesions of Mycosphaerella pinodes were often internally infected with the fungus. When infected seeds were sown in sterile grit almost all the seedlings showed disease lesions, at or below soil level, 4–6 weeks after sowing. Seed infected with Ascochyta pisi gave only 40% infection of seedlings: these showed lesions on the stem and first two leaves within 4 weeks of sowing. Infection of seeds by both pathogens could be eradicated by soaking the seeds for 24 hr. in 0.2% suspensions of thiram or captan at 30d?C. In laboratory or greenhouse tests these treatments did not check germination, but in the field the captan treatment reduced emergence. The treated seeds became fully imbibed but could be dried and stored: the thiram treatment was used for semi-commercial treatment of quantities of seed up to 3 cwt.     

Phytophthora cinnamomi: the Dynamics of Chlamydospore Formation and Survival

Chlamydospore activity was investigated by inoculating 10 day old seedlings of Lupinus angustifolius with Phytophthora cinnamomi. Infected roots were excised and buried in 4 non-sterile media: – glass beads, gravel, sand or soil at different water potentials and incubated at 22°C. Roots were examined and chlamydospores counted initially and after 10, 20 and 30 days. Large numbers of spores formed in the roots buried in the various media. Spore formation was rapid in glass beads and gravel, and slower in soil, but more chlamydospores survived the 30 day period when roots were buried in soil. Nylon mesh squares 1 cm²were inoculated with 38 chlamydospores and buried in the same 4 media. 70–100% of chlamydospores germinated and colonised 90–100% of the mesh, producing mycelia and an increased number of chlamydospores. In some instances there was a ten-fold increase in spore numbers and numbers were still increasing at 28 days. Maximum numbers and maximum survival occurred on mesh buried in soil. The role of the chlamydospores is considered, as that of a dynamic unit with a saprobic phase in the life cycle of the fungus which is independent of host, and has the capacity to increase both population density and distribution of the pathogen.     

Influence of pea-root exudates on germination of conidia and chlamydospores of physiologic races of Fusarium oxysporum f. pisi

Sterile root exudates from wilt susceptible and wilt resistant pea cultivars showed no differential effects on spore germination of Fusarium oxysporum Schl. f.pisi (Linf.) Snyd. & Hans, races 1 and 2 which could be correlated with the pathogenicity of a particular isolate to a given cultivar. Uniformly high percentages of germination were obtained with conidia of the two races in aseptic shake culture with exudates collected from resistant or susceptible plants of various ages. Chlamydospores of the two races incubated with exudates under sterile conditions germinated to uniformly high levels irrespective of exudate origin. Conidia and chlamydospores of Fusarium solani (Mart.) Sacc. f. pisi (Jones) Snyd. & Hans., used for comparative purposes, also germinated to high levels in the presence of exudate solutions of all cultivars. Non-specific germination of the two races of F. oxysporum f. pisi occurred in soil when the exudates were supplied to populations of chlamydospores via diffusion units. Germination was lower than that recorded under sterile conditions and was rapidly followed by germling lysis.     

Sporenkeimung von Phoma lingam (Tode ex. Fr.) Desm. und Resistenzprüfung bei Raps im Gewächshaus

Spore germination of Phoma lingam (Tode ex. Fr.) Desm. and methods to determine resistance of oil seed rape in the greenhouse It was the aim of this investigation to obtain more insight into the epidemiology of Phoma lingam (Tode ex. Fr.) Desm. (stat. gen. Leptosphaeria maculans (Desm.) Ces. et de Not.) and to improve the existing methods for resistance testing. In laboratory experiments the differing demands on temperature of both, the sexual as well as the non sexual phase were observed. Many ascospores developed germ tubes after eight hours at 4-8°C whilst pycnidiospores needed 24 hours at 16°C to have similar development. In greenhouse experiments young plants were infected by spraying or by placing a droplet of spore suspension onto cotyledons or leaves. Generally, ascospores were more virulent than pycnidiospores. The ascospores were obtained from old rape stalks which could be stored at -18°C without losing virulence. The most severe attack was observed after incorporating infested oat kernels (3 % w/ w) into soil, but the difference between cultivars vanished which was already low with the other methods, and which did not always correspond with results obtained in the field at stage 85, so that all these methods are not as suitable as those in the field. The distribution of pycnidiospores is also possible by adhering to the seed after threshing. The infection of the seedlings from this source was more pronounced in steamed than in unsteamed soil. The re-isolation of P. lingam increased as well from plants grown in steamed soil. Furthermore, pycnidiospores are distributed by wind during combining to neighbouring fields, already prepared at that time for rape sowing.     

Enhanced Recovery of Phytophthora ramorum from Soil Following 30 Days of Storage at 4°C

Chlamydospores of Phytophthora ramorum were used to infest field soil at densities ranging from 0.2 to 42 chlamydospores/cm³ soil. Recovery was determined by baiting with rhododendron leaf discs and dilution plating at time 0 and after 30 days of storage at 4°C, as recommended by USDA‐APHIS. Baiting was slightly more sensitive than dilution plating in recovering P. ramorum immediately following infestation of soil and allowed detection from samples infested with as little as 0.2 chlamydospores/cm³ compared with 1 chlamydospore/cm³ for dilution plating. After 30 days of infested soil storage at 4°C, P. ramorum was detected at significantly (P = 0.05) higher levels than at time 0 with both recovery methods. The results indicate that storage of P. ramorum‐infested soil at 4°C may allow for pathogen activity, such as sporangia production, which may enhance recovery from soil.     

Ascochitine Production by Fungi Responsible for Ascochyta Diseases of Pea

Ascochitine production by 24 isolates of three fungal species causing the Ascochyta disease complex in pea plants, was examined. All eight isolates of Ascochyta pisi Lib. secreted ascochitine to liquid nutrient medium in amounts of 0.1 to 1.2 mg/l. However, no ascochitine was found in culture filtrates of Mycosphaerella pinodes (Berk. and Blox.) Vestergr. or of Phoma medicaginis var. pinodella (Jon.) Boerema although the aggressiveness of these two species as pathogens was shown to be higher than that of A. pisi. Individual isolates of each of the three fungal species examined did not differ in their pathogenicity.     

Survival of Phytophthora cinnamomi in Buried Eucalyptus Roots

Colonization and survival of Phytophthora cinnamomi in roots was tested in 3 months old, axenically grown seedlings of Eucalyptus maculata (field resistant) and E. sieberi (susceptible). The roots were inoculated, then one week later were excised and buried in three non-sterile, conducive soils; a lateritic gravel, an infertile duplex soil, a loamy sand as well as in a fertile, suppressive krasnozem. Pathogen viability, percentage root colonization and chlamydospore numbers were examined at matric potentials of ?1/3, ?5 and ?10 bar after periods of 10, 100 and 200 days at 21°C. At 10 days, survival was 100% in the form of mycelium and the only significant difference was between the two Eucalyptus species. At 100 days survival was solely due to chlamydospores, but the pathogen was viable in all inoculated roots and at each matric potential. At 200 days soils had dried to less than ?10 bars and the pathogen failed to survive. No significant differences were found between the two pathogen isolates but significant differences were obtained between the susceptible and field resistant Eucalyptus species. Pathogen viability, percentage root colonization and chlamydospore number were highly correlated with soil types and matric potential. These components declined with decreasing soil matric potential. The Krasnozem was only suppressive at relatively high soil matric potentials (?1/3 bar). At lower values (?5, ?10 bar) survival of the pathogen, chlamydospore numbers and percentage colonization of the roots in the Krasnozem were comparable with that of the 3 conducive soils tested. Chlamydospores were present, but in low numbers in roots buried in the suppressive soil at ?1/3 bar.     

Winter survival of Ceramica pisi (Lepidoptera: Noctuidae) in Iceland

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Role of Inhibitors of Fungal Growth in the Limitation of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

The phytoalexin, pisatin, was detected in host tissues 24 hafter inoculation of pea leaflets with spores of the leaf-spottingpathogens Ascochyta pisi and Mycosphaerella pinodes. Pisatincontinued to accumulate in infected tissue as A. pisi lesionsdeveloped and was present in inhibitory concentrations in thebrown tissue beyond the region colonized by the pathogen. During the formation of limited M. pinodes lesions, concentrationsof pisatin were highest 2 days after inoculation. Levels weremore variable and lower in older lesions which appeared to containno other inhibitors of germ-tube growth. Spreading lesions causedby M. pinodes on leaflets floating on water contained littleor no pisatin although little was released to the water below.These lesions did, however, contain other highly active inhibitorsof germ-tube growth. The significance of these results in terms of limitation oflesions are discussed. The ease with which M. pinodes lesionscan become progressive may reflect the greater ability of thispathogen to grow in high concentrations of pisatin under certainconditions.     

The significance of temperature during sporulation on the biology of pycnidiospores of Mycosphaerella ligiilicola

The germination, infectivity and survival of pycnidiospores obtained from cultures of Mycosphaerella ligulicola grown at 15 and 26 °C were compared. Spores formed at 26° (‘26° spores’) were less able to germinate at low relative humidities and showed a narrower temperature range for maximum germination after 6 h. At high spore densities 26° spores showed self-inhibition of germination and, over a range of lower densities, growth of their germ tubes was checked, which resulted in lower infection of leaf discs compared with 15° spores in which this phenomenon did not occur. The fungus could be recovered from un-sterile compost over a longer period after inoculation with 15° spores. Only after storage at a temperature well below zero was there a difference in viability between 15° and 26° spores. It is thought that the potential advantage of producing larger numbers of spores at 26° would be realized only under optimum conditions for dispersal and infection. The smaller number of spores produced at 15° are likely to be successful under natural conditions.     

Endogenous arabitol and mannitol improve shelf life of encapsulated <Emphasis Type="Italic">Metarhizium brunneum</Emphasis>

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6?±?0.2 and 2.1?±?0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1?±?4.4 to 55.0?±?6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5?±?3.3% at 5 °C followed by 54.5?±?1.6% at 18 °C and 19.4?±?1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3?±?7.6 to 98.0?±?1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.     

Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

In vivo-Untersuchungen zur Entstehung und Funktion der Chlamydosporen von Botrytis cinerea Pers. am Wirt-Parasit-System Fuchsia hybrida – B. cinerea

In vivo-investigations on the formation and function of chlamydospores of Botrytis cinerea Pers. in the host-parasite-system Fuchsia hybrida–B. cinerea On naturally with Botrytis cinerea Pers. infected and artificially inoculated outdoor- and greenhouse-plants of Fuchsia hybrida the extra- and intramatrical formation of the B. cinerea- chlamydospores was investigated. The chlamydospores served 1. as structures of survival, which were tested with regard to their tolerance of drought, nutrient- and oxygen-deficiency, attack by bacteria and pH-requirements. 2. The chlamydospores represented dispersal units, which were capable of germination. 3. The chlamydospores could function as structures of infection, because after chlamydospore germination the outgrowing mycelium – either directly or after production of macroconidia – could serve as secondary inoculum and start new infections.     

Microcycle speculation in theClaviceps purpurea 244

The mutant strainClaviceps purpurea 244 forming hyphae composed mainly from sclerotiumlike cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 × 6.5 μm). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 μg/ml clavins, with elymoclavin predominating (82 %). The parent strainClaviceps purpurea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85 %) at a concentration of 4 mg/ml.