Influence of a Fungus-Feeding Nematode on Growth and Biocontrol Efficacy of Trichoderma harzianum

ABSTRACT A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80 degrees C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.     

Individual-based approach to modeling hyphal growth of a biocontrol fungus in soil

ABSTRACT Fungi in soil perform beneficial roles that include biological control of soilborne plant pathogens. However, relatively little predictive information is available about the growth and activity of fungal hyphae in soil habitats. A stochastic computer simulation model ("Fungmod") was developed to predict hyphal growth of the biocontrol fungus Trichoderma harzianum ThzID1 in soil. The model simulates a fungal colony as a population of spatially referenced hyphal segments, and is individual-based, in that records of spatial location and branching hierarchy are maintained for individual hyphal nodes. In this way, the entire spatial structure of the fungal colony (hyphal network) can be explicitly reconstructed at any point in time. Also, the soil habitat is modeled as a population of spatially referenced 1-mm(3) soil cells, allowing for the simulation of a spatially heterogeneous environment. Initial hyphal growth parameters were derived from previously published results, and the model was tested against new data derived from image analysis of hyphal biomass accumulation in soil. The ability to predict fungal growth in natural habitats will help to improve the predictability of successful myco-parasitic events in biological control systems.     

An appraisal of five methods for the measurement of the fungal population in soil treated with chlorothalonil

An experiment was carried out in a greenhouse with the aim of determining an acceptable method to quantify the fungal population in a soil treated with the fungicide chlorothalonil. Doses of the fungicide ranging from 12 to 96 µg AI g⁻¹ soil were applied and microbial biomass carbon (C), soil ergosterol content, living hyphal length, as well as counts of total and cellulolytic fungi colonies, were measured 1, 2, 3, 4 and 5 weeks after application. At the end of 16 weeks a new evaluation was done using the three first methods. The microbial biomass C was the least sensitive parameter and, on balance, the living hyphal length was the most sensitive parameter for demonstrating effects of chlorothalonil on the fungal population. Some problems related to the efficiency of the ergosterol content and living hyphal length measurements in the evaluation of the effect of the fungicide on the fungal microflora are discussed, as is the need to compare short‐term (0–5 weeks after treatment) with longer‐term (16 weeks) results. © 2000 Society of Chemical Industry     

Suppression of clubroot formation in Chinese cabbage by the root endophytic fungus, Heteroconium chaetospira

Root-colonizing fungi were isolated from a total of 663 root segments of Chinese cabbage plants grown in soils collected from wheat, rape, Chinese cabbage, and napier grass fields. Most of the 322 isolates were from the wheat field soil and comprised 18 genera and two septate fungal groups. Hyaline and dark septate fungi accounted for approximately half the isolates from the wheat field soil. Sixteen isolates almost completely suppressed clubroot in sterile soil. Amongst these 16 isolates, two from Heteroconium chaetospira were also effective in nonsterile soil. Chinese cabbage seedlings from seed treated with these two isolates appeared healthy, and inoculation with one isolate promoted growth. Hyphae of the fungus covered the root surface and extensively colonized the inner cortical tissues.     

Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil

Pelletized formulations of wheat bran or kaolin clay in an alginate gel containing conidia, chlamydospores, or fermentor biomass (FB) of several isolates of the biocontrol fungi Trichoderma spp. and Gliocladium virens were prepared. The ability of fungal propagules within the pellets to proliferate in soil was determined. Higher population densities were obtained when alginate pellets added to soil contained chlamydospores rather than condia, and bran rather than kaolin as the bulking agent. The active ingredient in pellets prepared from FB was approximately 5% biomass by weight and contained many chlamydospores. Colony-forming units (cfu) ranged from 10⁶'to 10¹⁰/g of soil after soil amendment with FB pellets of 12 Trichoderma and G. virens isolates. Population densities were high during the first 3 weeks of incubation and declined only gradually during 9 weeks. Propagules in FB pellets were more viable at 5° than at 25°C. Viability at 25°C remained high (> 70%) after 1 week, but declined to less than 10% after 24 weeks. Despite reduction in propagule viability in stored pellets, numbers of cfu formed after adding these pellets to soil were comparable with those formed from freshly prepared pellets.     

广西防城港首次截获苜蓿黄萎病菌

从一批进境的美国苜蓿草样品中保湿分离得到了3株与苜蓿黄萎病菌(Verticillium albo-atrum)相似的目标菌VA1、VA2、VA3.该目标菌在PDA上生长迅速,菌丝生长0.8～1.0 mm/d,2～3 d即可生成肉眼可见的菌落斑点;菌落为白色圆形,边缘规则呈圆形,菌丝较致密,气生菌丝较少,菌落中心乳白色奶冻状;培养4～5 d即后产生气生典型轮枝状分生孢子梗,7～8 d后菌落中央表面因产生休眠菌丝开始变成黑褐色至黑色,20 d后菌落的表面和背面大部分均变黑色,仍不产生微菌核和厚垣孢子.该目标菌的纯培养用V.albo-atrum特异引物Vaa1/Vaa2进行检测,PCR 扩增后得到预期330 bp的产物片段,产物序列与V.albo-atrum相应序列的相似性为100％.从而鉴定确认该目标菌为苜蓿黄萎病菌V.albo-atrum.     

Environmental effects on asexual reproduction in Arundo donax

Arundo donax L. (giant reed) is an invasive perennial plant that has spread widely in riparian areas in California, where it has altered wildlife habitats, created a fire hazard, compromised water conservation efforts, and affected flood control. Currently, physical removal is the primary means of controlling this weed, which is ineffective because of prolific asexual reproduction from an extensive rhizome system. We conducted controlled experiments on the sprouting potential of vegetative propagules, effects of storage duration and conditions on sprouting, and survival and growth of propagules in various soil types and moisture regimens. Sprouting and regrowth varied greatly with propagule type and size and with treatment and duration of storage after removal from the plant. Over 90% of stem and rhizome pieces with at least one node sprouted. Stem sprouting was affected by prior storage duration, temperature and moisture, whereas only storage duration and moisture affected rhizome sprouting. Sprouting was reduced by drying propagules at 30 °C for 1 week and by storage in a soil slurry. After 16 weeks, even propagules maintained optimally in moist soil showed reduced sprouting. Rhizome pieces sprouted readily from a soil depth of 25 cm, whereas stem pieces sprouted from less than 10 cm. Responsiveness of asexual reproduction in A. donax to environmental cues suggests that mechanical control can be improved by careful timing and treatment of cut biomass pieces to minimize or inhibit sprouting.     

The relative susceptibility of some cucurbits to an Iranian isolate of Phytophthora drechsleri

When zoospores of Phytophthora drechsleri were added to a hydroponic medium in which roots of cucurbit plants were immersed, marrow and pumpkin ( Cucurbita spp.) remained symptomless but cantaloup, melon and cucumber ( Cucumis spp.) wilted. Symptom development was proportional to the logarithm of day number after inoculation and approximately proportional to the logarithm of zoospore number in cantaloup and melon but not cucumber which was also less susceptible. The fungus became established preferentially in the hypocotyls of cantaloups. Ten times as many chlamydospores differentiated on infected cantaloup hypocotyls as on lupin roots when both tissues were buried in moist soil.     

Larval Bradysia impatiens (Diptera: Sciaridae) potential for vectoring Pythium root rot pathogens

A series of laboratory experiments were conducted to investigate the capacity of Bradysia impatiens (Johannsen) larvae to ingest propagules from two strains each of Pythium aphanidermatum (Edson) Fitzp. and P. ultimum Trow and transmit the pathogens to healthy geranium seedlings on a filter-paper substrate in petri dishes. The capacity of fungus gnat larvae to transmit P. aphanidermatum to seedlings rooted in a commercial peat-based potting mix and germination of Pythium oospores and hyphal swellings before and after passage through the guts of larval fungus gnats were also examined. Assays revealed that Pythium spp. transmission by larval fungus gnats varied greatly with the assay substrate and also with the number and nature of ingested propagules. Transmission was highest (65%) in the petri dish assays testing larvae fed P. aphanidermatum K-13, a strain that produced abundant oospores. Transmission of strain K-13 was much lower (<6%) in plug cells with potting mix. Larvae were less efficient at vectoring P. ultimum strain PSN-1, which produced few oospores, and no transmission was observed with two non-oospore-producing strains: P. aphanidermatum Pa58 and P. ultimum P4. Passage of P. aphanidermatum K-13 through larval guts significantly increased oospore germination. However, decreased germination of hyphal swellings was observed following larval gut passage for strains of P. ultimum. These results expand previous studies suggesting that larval fungus gnats may vector Pythium spp.     

Interactions of Pratylenchus thornei and Fusarium oxysporum f. sp. ciceris on Chickpea

ABSTRACT Fusarium oxysporum f. sp. ciceris and the root-lesion nematode Pratylenchus thornei coinfect chickpeas in southern Spain. The influence of root infection by P. thornei on the reaction of Fusarium wilt-susceptible (CPS 1 and PV 61) and wilt-resistant (UC 27) chickpea cultivars to F. oxysporum f. sp. ciceris race 5 was investigated under controlled and field conditions. Severity of Fusarium wilt was not modified by coinfection of chickpeas by P. thornei and F. oxysporum f. sp. ciceris, in simultaneous or sequential inoculations with the pathogens. Root infection with five nematodes per cm(3) of soil and 5,000 chlamydospores per g of soil of the fungus resulted in significantly higher numbers of propagules of F. oxysporum f. sp. ciceris with the wilt-susceptible cultivar CPS 1, but not with the wilt-resistant one. However, infection with 10 nematodes per cm(3) of soil significantly increased root infection by F. oxysporum f. sp. ciceris in both cultivars, irrespective of fungal inoculum densities (250 to 2,000 chlamydospores per g of soil). Plant growth was significantly reduced by P. thornei infection on wilt-susceptible and wilt-resistant chickpeas in controlled and field conditions, except when shorter periods of incubation (45 days after inoculation) were used under controlled conditions. Severity of root necrosis was greater in wilt-susceptible and wilt-resistant cultivars when nematodes were present in the root, irrespective of length of incubation time (45 to 90 days), densities of nematodes (5 and 10 nematodes per cm(3) of soil), fungal inocula, and experimental conditions. Nematode reproduction on the wilt-susceptible cultivars, but not on the wilt-resistant one, was significantly increased by F. oxysporum f. sp. ciceris infections under controlled and field conditions.     

拟粗壮螺旋线虫对棉花的致病力及其与棉枯萎病的复合症

 温室测定结果:拟粗壮螺旋线虫(Helicotylenchus pseudorobusius)接种量在每100cm³土壤100~1000条的条件下,接种30天后棉苗生长量比无线虫对照苗显著降低(P<0.05)。抗枯萎品种86-1在单接枯萎菌无线虫处理中不发生枯萎病,在枯萎菌(Fusarium oxysporum f. sp. vasinfectum)接种量为每克土7.5×10⁵孢子和线虫(每100cm³±500~1000条)组合处理中发病,棉苗枯萎病情指数与线虫接种量之间呈正相关(γ=0.97)。田间试验结果表明:防治线虫和枯萎病复合症用薰蒸剂棉隆每m²8~14g处理土壤比用非薰蒸性杀线虫剂克百威(呋哺丹),丙线磷(益收宝)和克线磷的效果更好。     

Artificial Infection of Rosellinia necatrix with Purified Viral Particles of a Member of the Genus Mycoreovirus Reveals Its Uneven Distribution in Single Colonies

ABSTRACT Rosellinia necatrix mycoreovirus 3 (W370) (RnMYRV-3/W370, described as RnMYRV-3 in this paper), a member of the newly established genus Mycoreovirus within the family Reoviridae, is the hypovirulence factor of the white root rot fungus, Rosellinia necatrix. Two virus-free fungal isolates (W37 and W97) that were somatically incompatible with the virus-harboring field isolate (W370) were transfected with purified RnMYRV-3 particles. Virus infection was confirmed by electrophoresis and northern hybridization of viral double-stranded RNA. RnMYRV-3 was transmissible from transfected strains to their respective, virus-free counterparts via hyphal anastomosis. Virus-transfected strains produced smaller lesions on apple fruits than did their virus-free counterparts. Virus-cured strains were indistinguishable from wild-type strains in culture morphology and displayed approximately the same virulence level on apples. Virus-transfected strains had "mosaic" colony portions consisting of thin, fast-growing and dense, slow-growing mycelia, and grew more slowly as a whole than their virus-free, parental strains. The level of virus accumulation varied among virus-transfected subcultures and within its single colonies. Virus-transfected strains were occasionally cured, as was W370. Such a phenomenon may be ascribed to uneven viral distribution in single colonies and the difficulty in viral transmission to virus-free hyphae.     

The effect of seven substituted urea herbicides on the soil microflora

Chloroxuron, diuron, fluometuron, metobromuron and monuron added to soil at 500 parts/million a.i. on a dry weight basis caused an initial stimulation of CO₂ production, followed by indications of inhibition. Nitrification was clearly inhibited, particularly by monuron and metobromuron. Metoxuron at 5 and 50 parts/million a.i. had no effect on CO₂ output and nitrification, but at 500 parts/million a.i. both were greatly reduced. Numbers of fungal propagules were temporarily lower at 50 parts/million a.i., and severely curtailed at 500 parts/million a.i. but total counts of bacterial propagules were greatly increased. Linuron had an inhibitory effect on CO₂ evolution at 500 parts/million a.i. but this varied in extent and duration; some reduction was also found at 50 parts/ million a.i. At 500 parts/million a.i. there was no effect on mineralisation of N but in soil supplemented with ammonium sulphate nitrification was reduced. Changes in microbial equilibrium occurred, but were less marked than with metoxuron. Cellulose decomposition was also reduced. Small amounts of 3,4dichloroaniline were found in linuron-treated soils, but microbial activities were not affected when 5 parts/million 3,4-dichloroaniline was added to the soil. However, at 140 parts/million microbial activities were affected adversely, but in a different pattern to that for linuron; nitrification was severely reduced, but numbers of fungal propagules were hardly affected, suggesting that the effects observed in linuron-treated soil were not due to this metabolite.     

Effect of soil matric water potentials on germination of ascospores of Monosporascus cannonballus and colonization of melon roots by zoospores of Olpidium bornovanus

Results document, for the first time, the role of soil moisture on a unique, tripartite, host-specific rhizosphere interaction (i.e., Cucumis melo-Monosporascus cannonballus-Olpidium bornovanus). Specifically, colonization of cantaloupe roots by zoospores of O. bornovanus and germination of ascospores of M. cannonballus were highest at a soil matric potential of ?0.001 MPa but significantly inhibited at a matric potential of only ?0.01 MPa. Matric water potentials of ?0.01 MPa or drier are characteristically inhibitory to the motility of zoosporic microbes but not hyphal growth of filamentous fungi like M. cannonballus. These results support our previous conclusion that germination of ascospores of M. cannonballus, a destructive root pathogen of cantaloupe is mediated by O. bornovanus, an obligate, zoosporic fungus.     

The detection and estimation of Verticillium dahliae in naturally infested soil

The effects of several factors on the estimation of Verticillium dahliae in soil by the wet-sieving method were studied. The following factors were important for maximising recovery: the removal of soil particles of < 20 μm size from suspensions before plating; the medium used for plating; the amount of sieved soil inoculated to plates; the length of time of incubating plates; and the method of incubating soil on plates. There was no short-term effect of air-drying soil before analysis. Using sodium hexametaphos-phate to aid soil dispersion, treating soil suspensions with sodium hypochlorite, or making suspensions from different amounts of soil had no consistent effect on recovery. Excessive sample-to-sample variability in the results of some experiments was attributed to non-random distribution of fungus propagules in soil, or to the difficulty of standardizing the sieving part of the analysis, or both. Wet-sieving was generally more effective in detecting V. dahliae and gave higher counts than a sucrose-flotation method. Bait methods with eggplants or antirrhinums proved ineffective for estimating V. dahliae in naturally infested soil.     

Impact of Brassica juncea biofumigation on viability of propagules of pernicious weed species

Biofumigation may be a promising tool for depletion of persistent weed seedbanks/bud banks. This technique is based on the incorporation of chopped glucosinolate‐rich plant biomass into the soil, upon which isothiocyanates with herbicidal properties are released. To gain acceptance by farmers and foster its implementation, the biofumigation process should be further optimised. This study elucidated the impact of biological (species), technical (burial depth, ground cover) and pedohydrological (temperature and moisture content) factors on efficacy of Brassica juncea biofumigation under (semi‐)natural conditions. In a first experiment (field experiment), seeds and vegetative propagules of various weed species were buried at different depths and exposed to different doses of fresh fine‐chopped B. juncea biomass in the presence or absence of a plastic ground cover. In a second experiment (container experiment), buried seeds of ten species were subjected to biofumigation at diverging soil organic matter content, soil moisture content and soil temperature. In a third experiment (dose–response Petri dish bioassay), unburied seeds of eight species were subjected to various doses of rehydrated B. juncea powder. Biofumigation efficacy was determined by analysing viability of treated and untreated propagules. In general, efficacy of biofumigation increased with decreasing burial depth and increasing B. juncea dose. Biofumigation was highly effective (mortality >85%) against small‐seeded species but less effective (mortality 0%–20%) against hard‐seeded and large‐seeded species at 200 t ha^?1. Vegetative propagules of Sonchus arvensis, Equisetum arvense and Convolvulus sepium were highly sensitive (mortality >90%) to biofumigation. Efficacy was most pronounced under moist warm incubation conditions, in the presence of a plastic ground cover.     

Correlations between most probable number and activity of nematode-trapping fungi

ABSTRACT Soil cages were used to determine whether nematode-trapping fungi population density, as measured by most probable number (MPN) procedures, was correlated with the trapping of nematodes. Fungi studied (and trap type) were Arthrobotrys oligospora (adhesive networks), A. eudermata (adhesive networks), A. dactyloides (constricting rings), Dactylellina ellipsospora (adhesive knobs), and D. haptotyla (adhesive knobs). The fungi were formulated as assimilative hyphae in dried alginate pellets. Pellets were added to field soil, the soil was packed into 80-cm(3) cages (PVC pipe, 3.0 cm long and 3.9 cm in diameter), and the cages were buried in vineyards. After 14 to 61 days, the cages were recovered, and MPN data and trapping activity were determined. For all five fungi, MPN data were correlated with the number of pellets added. Regardless of fungus population density, A. oligospora and A. eudermata trapped few if any nematodes in soil, and consequently, trapping and fungus population density were not correlated. The correlation between population density and trapping was weak for A. dactyloides but relatively strong for D. ellipsospora and D. haptotyla. High levels of trapping by the latter two fungi required more than 10(2) fungus propagules per gram of soil.     

Effect of Water Potential on Conidial Germination and Antagonism of Ulocladium atrum Against Botrytis cinerea

ABSTRACT The saprophytic fungus Ulocladium atrum was selected for its ability to competitively exclude Botrytis spp. from aboveground necrotic plant tissues which can play a crucial role in the epidemiology of diseases caused by necrotrophic Botrytis spp. Fungal growth in necrotic aboveground tissues can be hampered by fluctuating water availability. Adaptation to these adverse conditions is a key factor for the successful establishment of an antagonist population in this niche. Conidia of U. atrum germinated at water potentials between -1 and -7 MPa on water agar. Germinated conidia survived one to three interruptions of moist incubation by periods at -10 or -42 MPa. The speed of germination was significantly slower with interruption of the moist period as compared with the control, which had continuously moist incubation. However, the maximum germination percentage was the same for conidia incubated with or without interruption of the moist incubation. In bioassays with necrotic cyclamen leaves at -1, -3, and -7 MPa, U. atrum significantly reduced the sporulation of B. cinerea by more than 80%. The results demonstrate that U. atrum tolerates water stress during competitive substrate colonization with B. cinerea. The antagonist is, therefore, an attractive candidate for field applications on aboveground tissues.     

Differential interactions of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> with <Emphasis Type="Italic">Brassica napus</Emphasis> detected with molecular and histological techniques

The differential interactions of V. longisporum (VL) and V. dahliae (VD) on the root surface and in the root and shoot vascular system of Brassica napus were studied by confocal laser scanning microscopy (CLSM), using GFP tagging and conventional fluorescence dyes, acid fuchsin and acridin orange. VL and VD transformants expressing sGFP were generated by Agrobacterium-mediated transformation. GFP signals were less homogenous and GFP tagging performed less satisfactory than the conventional fluorescence staining when both were studied with CLSM. Interactions of both pathogens were largely restricted to the root hair zone. At 24 h post-inoculation (hpi), hyphae of VL and VD were found intensely interwoven with the root hairs. Hyphae of VL followed the root hairs towards the root surface. At 36 hpi, VL hyphae started to cover the roots with a hyphal net strictly following the grooves of the junctions of the epidermal cells. VL started to penetrate the root epidermal cells without any conspicuous infection structures. Subsequently, hyphae grew intracellularly and intercellularly through the root cortex towards the central cylinder, without inducing any visible plant responses. Colonisation of the xylem vessels in the shoot with VL was restricted to individual vessels entirely filled with mycelium and conidia, while adjacent vessels remained completely unaffected. This may explain why no wilt symptoms occur in B. napus infected with VL. Elevated amounts of fungal DNA were detectable in the hypocotyls 14 days post-inoculation (dpi) and in the leaves 35 dpi. Root penetration was also observed for VD, however, with no directed root surface growth and mainly an intercellular invasion of the root tissue. In contrast to VL, VD started ample formation of conidia on the roots, and was unable to spread systemically into the shoots. VD did not form microsclerotia in the root tissue as widely observed for VL. This study confirms that VD is non-pathogenic on B. napus and demonstrates that non-host resistance against this fungus materializes in restriction of systemic spread rather than inhibition of penetration.     

梅奇酵母XY201对冬枣采后病原真菌的抑制效果

本文对梅奇酵母Metschnikowia zizyphicola XY201进行了生物量最大值培养时间、不同环境条件下的活力变化及其对冬枣采后病原真菌抑制效果的研究。结果表明,梅奇酵母XY201菌株在NYDB培养基中培养96h后,菌体生物量达最大值3.84×108cfu/ml。XY201在20℃、4℃和0℃三个不同温度和不同介质条件下的生活力变化不同,0℃培养液菌体生活力最强,240d时菌液浓度为6.46×107cfu/ml。XY201能显著抑制冬枣采后病原菌扩展青霉Penicillium expansum、细极链格孢Alternaria alternata、黑曲霉Aspergillus niger和黑根霉Rhizopus nigricans的孢子萌发和菌丝伸长;当酵母菌浓度为5×108cfu/ml时,对病原菌的抑制率可达100%。对峙培养结果显示XY201对A.alternata生长的抑制率为42.59%。