Improved selective media for isolation ofTrichoderma spp. orFusarium spp.

Modifications were made to improve theTrichoderma selective medium (TSM). TSM supplemented with benomyl was efficient for isolatingFusarium spp. from soil; and TSM supplemented with captan was a specific selective medium forTrichoderma spp., even in the presence ofFusarium in the soil.     

A selective medium for isolation and identification ofBotrytis spp. from Soil and onion Seed

The population ofBotrytis allii in either naturally or artificially infested soils was selectively measured by the soil dilution plate count procedure on a developed synthetic medium supplemented with tannic acid, fungicides, antibiotics and Cu⁺⁺ions. Conversion of tannic acid into a dark brown pigment was related to the activity of extracellular polyphenoloxidase produced by the fungus. Thus, brown-pigmented colonies were recognized asBotrytis spp. The same medium was used for detecting the presence of the fungus on onion seed.     

An improved selective medium for the isolation ofVerticillium dahliae

A selective medium was developed for the isolation and enumeration ofVerticillium dahliae in senescent infected tomato tissues heavily colonized with various fungi, from which the pathogen could not be isolated by the common ethanolstreptomycin selective medium. Suppression of the accompanying saprophytes, especiallyFusarium, was achieved by the combined effects of relatively low incubation temperature (18°C) and supplementation of the medium with ethanol (0.5%), pentachloronitrobenzene (50 ppm) and antibiotics. Addition of sucrose (0.75%) and Czapek’s salts improved visualization ofV. dahliae colonies which appeared black.V. dahliae populations were estimated in senescent infected tissues from various sources by use of this medium.     

Control by metham-sodium ofxanthomonas campestris pv.Campestris and the pathogen’s survival in soil

Detection and quantitative estimation ofXanthomonas campestris pv.campestris (XCC) from soils were done on Kritzman’s selective medium (KSM). The composition of KSM, its selectivity and the recovery of 64 isolates ofXCC obtained from Israel, Holland, Taiwan, the USA, and Germany are given in detail.XCC was isolated from soil in Israel 22 months after infected cauliflower residues were incorporated into the soil. Metham-sodium (MES) at 16 ml/kg (32.7% active compound) of air-dried soil killed more than 99.9% and 99% ofXCC in soil in laboratory and field tests, respectively. MES applied at this ratevia the irrigation system in a field infested withXCC, significantly reduced counts of the pathogen in stems, siliques and seeds, and the incidence of plants with black rot symptoms. The treatment was more successful in reducing disease incidence when applied to the soil at a depth of 30 or 40 cm rather than 20 cm. It is recommended that a seed treatment be combined with fumigation of soil with MES for the production of seeds free ofXCC.     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Effect of label and sublabel rates of metam sodium in combination with<Emphasis Type="Italic">Trichoderma hamatum,T. harzianum,T. virens,T. viride</Emphasis> on survival and growth of<Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

This work was undertaken to determine the effects ofTrichoderma spp. combined with label and sublabel rates of metam sodium on survival ofRhizoctonia solani in soil. Soils were infested with wheat bran preparations ofTrichoderma hamatum Tri-4,T. harzianum Th-58,T. virens Gl-3, andT. viride Ts-1-R3. Soil was also infested with sterile beet seeds that were colonized withR. solani. Beet seeds were later recovered, plated onto water agar plus antibiotics, and the growth ofR. solani was recorded. Preliminary experiments showed thatT. hamatum andT. virens reduced survival and saprophytic activity ofR. solani when the biocontrol fungi were incorporated into soil at 1.5% (w:w) or greater. Based on these data, biocontrol fungi in subsequent experiments were incorporated into soil at 2%. Metam sodium at label rate killed all biocontrol fungi andR. solani. At 1:2 and 1:5 dilutions, metam sodium reduced survival ofR. solani and allTrichoderma spp. When biocontrol fungi plus the label rate of metam sodium and 1:5, 1:10, 1:50 or 1:100 dilutions of the label rate were tested together, there were no interactions between any biocontrol agent and the fumigant with respect to colony diameter, reflecting that allTrichoderma isolates tested reacted similarly to increasing concentrations of metam sodium. At the label rate of metam sodium, allTrichoderma spp. significantly reduced colony diameter, but not growth rate, ofR. solani from beet seed. For the levels of metam sodium tested in combination withTrichoderma, it does not appear feasible to use a reduced rate of metam sodium to controlR. solani. However, the combination ofTrichoderma with metam sodium does reduce growth ofR. solani in comparison with that provided by metam sodium at the label rate. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Integrated control of soilborne and bulbborne pathogens in Iris

Coating iris bulbs with a preparation ofTrichoderma harzianum was highly effective under greenhouse conditions in reducing incidence of diseases caused byRhizoctonia solani andSclerotium rolfsii. In field experiments with irises for bulb production, the incidence ofR. solani in plants and bulbs was effectively reduced (up to 93%), and the yield increased (by 35–41%), by applyingT. harzianum either as a bulb coating or broadcast application (biological treatment), treating soil with pentachloronitrobenzene (PCNB; quintozene) (chemical treatment), or solarizing the soil by mulching it with transparent polyethylene sheets (physical treatment) prior to planting. Combined treatments,i.e., chemical-biological or physicalbiological, were the most effective.T. harzianum bulb treatment and broadcast application in field plots increasedTrichoderma population density in the soil by 4–27 times.     

Cellulose-decomposing fungi in polder soils and their possible influence on pathogenic fungi

For a study of the colonisation of the IJsselmeerpolders by fungi, attention was fixed on the cellulose-decomposing fungi, since these are considered to represent the more stable part of the mycoflora of soils. If one assumes that in the North East Polder, cultivated for approximately 25 years, the fungal population is approaching stability, the progress of colonisation of the younger polders can be judged by comparison of the number of species found and their frequency.It is shown that the progress of fungal colonisation did not depend only on the time during which a soil has been dry, but also on the use that has been made of it. Of the species known as antagonists of other fungi,Trichoderma spp. provided an increasingly larger proportion of the total population as the soils were further from stability.It is concluded that the increase of fungal antagonists may well be one of the causes of the decline of such parasites asOphiobolus graminis andRhizoctonia solani. Special mention is made ofGliocladium roseum, the parasitism of which towardsR. solani could be demonstrated in paired cultures.     

Selective accumulation of Trichoderma species in soils suppressive to radish damping-off disease after repeated inoculations with Rhizoctonia solani, binucleate Rhizoctonia and Sclerotium rolfsii

Artificial suppression of radish damping-off disease was induced by repeated soil inoculations with Rhizoctonia solani, binucleate Rhizoctonia (BNR) and Sclerotium rolfsii in pot systems. Soils repeatedly inoculated with R. solani and BNR showed suppressive to disease caused by R. solani and S. rolfsii, while soils repeatedly inoculated with S. rolfsii were suppressive to disease caused by S. rolfsii but not by R. solani. Species of Trichoderma were consistently isolated from soils repeatedly inoculated with R. solani, BNR and S. rolfsii. These Trichoderma spp. accumulated selectively in relation to the fungal species that was repeatedly added to the soils. The ratios of the frequencies of T. viride, T. harzianum and T. hamatum were 5:2:2 and 8:5:2 in soils repeatedly inoculated with R. solani and BNR, respectively. In S. rolfsii- inoculated soils, T. koningii was predominantly isolated. T. viride, T. harzianum and T. hamatum isolates obtained from either R. solani or BNR after repeated additions to the soils suppressed radish damping-off disease caused after challenge inoculations with R. solani or S. rolfsii. Among the Trichoderma species, T. viride consistently yielded high levels of suppression. However, isolates of T. koningii obtained from S. rolfsii-infested soils suppressed disease caused by S. rolfsii but failed to suppress disease caused by R. solani. Generally, the species of Trichoderma accumulated in a selective pattern that was closely related to the species of fungal pathogen used to induce the suppressive soil.     

Species of Pythium in the Netherlands

A review is given on observations ofPythium spp. so far published. In personal investigations about 4000 isolates ofPythium species were obtained from soil, water, Daphnias and plants during 1966–1972. Special attention was paid to the progressive colonization of the newly reclaimed polder Zuidelijk Flevoland: the numbers of species per sample increased from 0 in 1966 to 13 in 1972. The percentage of samples which did not contain anyPythium decreased from 54 in 1968 to 0 in 1972. From Zuidelijk Flevoland altogether 21 species were isolated, from Oostelijk Flevoland 22, of which 19 occurred in both the polders. The soil depth down to 10 cm had little influence on the occurrence ofPythium species. From soils near Rhenen and Zutphen 12 and 19 species respectively were isolated, from flax and flaxfields 11, from forests 8, from roots of different plants 17 and from water 11. Besides species already previously recorded, 15 new records ofPythium species for the Netherlands are documented. Species with filamentous non-swollen sporangia preferentially occurred in water and wet soils while species with spherical sporangia or hyphal swellings dominated in cultivated soils, forests and in the roots of phanerogams. In 1968 species with filamentous non-swollen sporangia comprised 89% of the isolations from Zuidelijk Flevoland; this percentage decreased to 9 in 1972. The percentage of isolates with spherical sporangia or hyphal swellings from cultivated soils, forest soils or plant material was mostly over 90.P. rostratum Butler, though found in all kinds of soils, was often the only species which could be isolated from dry sandy soil samples.P. sylvaticum Campbell & Hendrix was the most common species in cultivated soils in the Netherlands (37% of the isolates in 1968–1972), followed byP. oligandrum Drechsler (22%) andP. paroecandrum Drechsler (11.5%).     

Detection of Clavibacter michiganensis ssp. michiganensis in tomato seeds by immunofluorescence microscopy and dilution plating

A method for detectingClavibacter michiganensis ssp.michiganensis in tomato seeds was evaluated. The method is based on rapid screening of tomato seed lots using indirect immunofluorescence staining (IF), followed by dilution plating of IF positive seed lots. Different polyclonal antisera, prepared againstC. michiganensis ssp.michiganensis were tested for their specificity using IF. All strains ofC. michiganensis ssp.michiganensis tested reacted with the polyclonal antisera. Two of nine saprophytic isolates from tomato seeds were positive with the antisera as well as with the control normal serum, but cells of these isolates were distinct in shape from cells ofC. michiganensis ssp.michiganensis.For extraction of the pathogen from the seed, seeds were either blended with a stomacher or soaked at 4–6 °C. The stomacher method yielded more fluorescent cells in IF than 24 h soaking of seed samples. However, soaking of seeds for 48 h generally yielded less saprophytes and overall higher numbers ofC. michiganensis ssp.michiganensis colonies in dilution plating when compared to blending by a stomacher. SCM medium was generally more selective than KBT and modified CNS medium. However, the efficacy of the medium was dependent on the seed lot and/or extraction method used. Confirmation of suspected colonies with YDC (yeast-dextrose-carbonate medium), IF and a pathogenicity test on tomato seedlings proved to be highly reliable (P>0.95). For routine testing of seed lots it is recommended to screen tomato seed lost after soaking seeds for 24 h at 4–6 °C with IF, followed by plating of IF-positive seed lots on modified CNS and SCM after soaking seeds for an additional 24 h.     

A selective agar medium for isolation,enumeration and morphological identification of Fusarium proliferatum

A selective agar medium based on macerated date fruits was developed for the isolation, enumeration and morphological identification of Fusarium proliferatum from soil and from infected tissues of various plants (including: onion bulbs, corn ears and stems, and various weed tissues). The selective date medium enhances the formation of polyphialide and longer chains of conidia for better separation from other related Fusarium species which also grow and proliferate on this medium. Furthermore, the date medium enables microscopic distinction among other closely related Fusarium species, e.g. F. oxysporum and F. verticillioides. Fruits of the date cultivars Medjoul and Deglet Noor provided the most useful results as compared with other cultivars tested. The date medium can serve as a selective medium for direct isolation and enumeration of F. proliferatum, as it suppresses the development of other soil fungi and plant pathogens such as Macrophomina phaseolina, Sclerotium rolfsii and Rhizoctonia solani, as well as bacteria.     

Growth and survival of the mycoparasiteConiothyrium minitans on lettuce leaves in contact with soil in the presece or absence ofSclerotinia sclerotiorum

Lettuce leaves co-inoculated withSclerotinia sclerotiorum andConiothyrium minitans and controls were placed on, or buried in, soil for a period of two weeks to study development and survival ofC. minitans. OnS. sclerotiorum-infected leaves on the soil surface, the number of colonies ofC. minitans recovered was about 40% of the number of pycnidiospores applied. When buried in the soil there was a reduction to about 2% of the spores applied. WhenC. minitans was applied on healthy lettuce leaves, which were subsequently placed on or in soil, the recovery was about 4%. It is argued that these figures indicate multiplication ofC. minitans onS. sclerotiorum-infected lettuce leaves on the soil, and good survival in all other cases.     

Evaluation of selective media and bait methods for estimating Phytophthora cactorum in apple orchard soils

Soil plating, with a specially devised selective medium, gave estimates of Phytophthora cactorum in an East Mailing Research Station apple orchard soil up to three times those obtained by dilution and baiting with apple seedlings or cotyledons and using the most probable number analysis.
When the same techniques were applied to a range of soils from apple orchards in south-east England with a history of P. cactorum diseases the plating method failed in most instances, mainly because Pythium spp. rapidly swamped the plates. The dilution/baiting method was applicable to all soils though there was a tendency to underestimate because of anomalous results at lower soil dilutions.
Oospores were the only propagules which could be confirmed as sources of P. cactorum colonies on soil isolation plates.     

Detection,quantification and classification of soft rot erwinias associated with potato tubers

An improved method for detection, quantification and classification of soft rot bacteria associated with potato seed tubers, plant material, soil and water has been developed. The method is based on the use of a modified version of the crystal violet pectate selective medium (CVP), enrichment cultures under anaerobic conditions using pectate as the sole carbon source for recovery improvement, and the quantitative estimation ofErwinia spp. by employing a new solid medium - most probable number (MPN) method. The use of this method enabled an improvement in the recovery and identification of specificErwinia spp. in mixed populations. This was done by incubating CVP plates — used for the MPN counting — at three different temperatures (15, 28 and 39°C). These combined techniques were used for estimating low level populations at less than one cell per gram or ml tested ofErwinia carotovora subsp.carotovora, E. carotovora subsp.atroseptica, andE. chrysanthemi.     

Suppression of Rhizoctonia solani in potato fields. II. Effect of origin and degree of infection with Rhizoctonia solani of seed potatoes on subsequent infestation and on formation of sclerotia

The seed potatoes used in these experiments had been grown in a slightly acid pleistocene sandy soil or in a marine, holocene sandy loam. They were free of sclerotia ofR. solani or lightly or moderately speckled with them. Seed potatoes from the sandy soil produced plants that suffered less fromRhizoctonia than plants from seed potatoes that had been grown on the marine sandy loam. Similarly harvested tubers had, in a non-conducive soil and in conducive soils with a (very) low inoculum density ofR. solani, fewer sclerotia when they came from seed potatoes grown in an acid sand. In each soil, the degree of infestation of the crop not only depended on the severity of infection of the seed potatoes, but also on their origin. With regard to sclerotia production on tubers, three types of soil were distinguished: suppressive, conducive with a high, and conducive with a very low inoculum density ofR. solani. The differences in infestation and in the amounts of sclerotia on tubers between the crop grown from seed potatoes from the sandy soil and that from seed potatoes from the marine sandy loam soil, is attributed to a richer load of antagonists on the former and possibly to a larger proportion of saprophyticRhizoctonia strains among their sclerotia. The antagonists seem to be inhabitants of the subterranean parts of the plant and to function independently of the soil. This implies possibilities for their use in biological control.     

A selective medium to isolate airborne spores of Microdochium nivale,causing winter wheat scab

Winter wheat scab in Hokkaido, Japan is caused predominantly by Gibberella zeae and Microdochium nivale and can result in significant yield losses. A selective medium for isolation of G. zeae was previously developed, but not for M. nivale. The purpose of this study therefore was to develop a selective medium for isolation of airborne spores of M. nivale. Based on the basic composition of Komada’s Fusarium-selective medium, carbon and nitrogen sources and the most suitable vitamin B component for the basal composition were examined. Hyphal growth of M. nivale was promoted when galactose was replaced with lactose and combined with L-asparagine, while aerial hyphal formation increased with thiamine hydrochloride as the vitamin B source. In antimicrobial composition, colony formation of other filamentous fungi was greatly inhibited by spiroxamine. Thiophanate methyl, to which M. nivale shows resistance, selectively inhibited the growth of Fusarium spp. only. Spore trapping using the selective medium was subsequently performed in a wheat field. M. nivale formed characteristic pinkish colonies on the selective medium in the case of contamination with other filamentous fungi, making differentiation easy. Overall, the findings show that LATTS medium developed in this study is effective for isolation of airborne spores of M. nivale.     

Sensitivity to chloropicrin of microsclerotia ofVerticillium dahliae as related to their germination on growth media and in soil

Sensitivity of microsclerotia ofVerticillium dahliae to chloropicrin increased considerably during incubation on Czapek’s medium and in moist soil. However, whereas on a growth medium maximum sensitivity was obtained after 48–96 h, a period of 216–288 h was required to obtain a similar degree of sensitivity of microsclerotia incubated in moist soil. At the end of the incubation period, the microsclerotia which had been incubated in moist soil gave rise to an average of four hyphae per microsclerotium as compared with 130 observed on Czapek’s agar. It is concluded that germination does not play a major role in the increase of sensitivity to chloropicrin during the incubation of microsclerotia ofV. dahliae in moist soil.     

Use of a selective medium to study the dispersal of ascospores ofSclerotinia sclerotiorum

A selective medium (potato dextrose agar with pentachloronitrobenzene and streptomycin) was used to assess dispersal of ascospores ofSclerotinia sclerotiorum in the field. This medium inhibited development ofMucor spp. andRhizopus spp. without affecting the germinability ofS. sclerotiorum ascospores or the production of sclerotia. The number of viable ascospores started increasing in December, was maximal from January to February, and decreased sharply in March. Deposition of ascospores on plates was maximal (57%) between 10:00 and 13:00 hours. Seventy-seven to 90% of the ascospores were deposited within the first 100 m from the source of inoculum and the rest were transported farther.     

Suppression of potato cyst nematode root penetration by the endoparasitic nematophagous fungusHirsutella rhossiliensis

The endoparasitic nematophagous fungusHirsutella rhossiliensis was tested for its ability to suppress root penetration and cyst formation by the potato cyst nematode speciesGlobodera pallida. Isolates ofH. rhossiliensis were obtained from infected potato cyst nematode juveniles from different starch potato fields in The Netherlands. The isolates showed no difference in spore adhesion to juveniles on agar plates (adhesion rate: ±90%). The most rapid growing isolate, CBS 108.94, was used for experiments. Vegetative mycelial colonies ofH. rhossiliensis CBS 108.94, grown in potato dextrose broth, were used as soil inoculum. During submerged cultivation the mycelial colonies produced phialides (spore-bearing cells) but no spores. Exposed to the air, however, spores were rapidly formed. The effect of different soil inoculum densities of mycelial colonies on root penetration byGlobodera pallida was examined in an experiment in 250-ml pots. Up to a mycelial colony concentration representing a potential spore density of 10⁴ g^–1 soil no suppression occurred. At approximated densities of 2.5×10⁴ and 10⁵ spores g^–1 soil the numbers of juveniles which penetrated roots were reduced by 30% and 34%, respectively. The distribution of the inoculum could be improved by fragmentation of the mycelial colonies before soil inoculation. Using mycelial fragments, again no suppression of root penetration was observed up to a potential spore density of 10⁴ g^–1 soil, but at densities of 10⁵ and 10⁶ g^–1 a suppression of 54% and 88%, respectively, was measured. In a greenhouse experiment, soil inoculation with mycelial colonies with a potential spore production of 2.5×10⁵ g^–1 soil resulted in a suppression of root penetration of 37% and 51% after 5 and 6 weeks, respectively, but the number of newly formed cysts after 18 weeks in soil was not different for control and inoculated pots. It is concluded thatH. rhossiliensis may be useful for the reduction of root damage caused by juveniles of potato cyst nematodes, but the usefulness for population control is doubtful.