Selection of fungal biological control agents of Sclerotium cepivorum for control of white rot by sclerotial degradation in a UK soil

A range of fungal isolates was tested in a three-stage screening system for their ability to degrade sclerotia of Sclerotium cepivorum on agar and in soil, and to reduce white rot disease on onion seedlings. Biological control agents (BCAs) were identified that could degrade up to 60% of sclerotia in soil and significantly reduce white rot disease on onion seedlings. The efficacy of the BCAs was enhanced when applied as wheat bran cultures compared with spore suspensions, and two of the best BCAs from the screening procedures were both identified as Trichoderma viride (L4, S17A). When L4 and S17A were fluid-drilled in guar gum with bulb onion seed in the field white rot symptoms were significantly reduced, but stem base applications applied mid-season had little effect. The strategy of selecting and using BCAs that degrade sclerotia of S. cepivorum and integration with other control methods is discussed.     

Allium White Rot Suppression with Composts and Trichoderma viride in Relation to Sclerotia Viability

ABSTRACT Allium white rot (AWR) is a serious disease of Allium spp. caused by the sclerotium-forming fungus Sclerotium cepivorum. This work has examined the effects of onion waste compost (OWC) and spent mushroom compost (SMC), with and without Trichoderma viride S17A, on sclerotia viability and AWR in glasshouse and field experiments. Incorporation of OWC into soil reduced the viability of sclerotia and the incidence of AWR on onion plants in glasshouse pot bioassays, whereas SMC or T. viride S17A only reduced incidence of AWR. In two field trials, OWC reduced sclerotia viability and was as effective in reducing AWR as a fungicide (Folicur, a.i. tebuconazole). Field application of SMC had no effect on sclerotia viability and did not control AWR. However, the addition of T. viride S17A to SMC facilitated proliferation of T. viride S17A in the soil and increased the healthy onion bulb yield. The results indicate two mechanisms for the suppression of AWR: (i) reduction in the soil population of viable sclerotia, which may be due to volatile sulfur compounds detected in OWC but absent in SMC, and (ii) prevention of infection of onion plants from sclerotia following amendment of soil with OWC, SMC, or T. viride S17A.     

Integrated control of Allium white rot with Trichoderma viride, tebuconazole and composted onion waste

Two isolates of Trichoderma viride (L4 and S17A) were assessed for biological control of Allium white rot (AWR) with different onion accessions and cultivars, alone and in combination with a tebuconazole-based seed treatment or composted onion waste. In glasshouse tests, 23 new bulb-onion accessions from previous work to detect resistance to Sclerotium cepivorum showed no differences in susceptibility to AWR but, when combined with S17A, disease was reduced by up to two-thirds over all accessions. Trichoderma viride L4 and S17A also reduced the proportion of infected plants for five commercial bulb-onion cultivars and one advanced breeding line by at least one-third. Further glasshouse tests using a salad-onion cultivar showed that L4, S17A, tebuconazole or composted onion waste controlled AWR and at least halved the proportion of diseased plants. Combination treatments of T. viride with either tebuconazole or compost enhanced control and, in some treatments, disease was almost eliminated. In field trials, control of AWR by S17A was significant for 17 out of 18 individual or mixed bulb-onion accessions, with disease reduced overall by more than half. In another field experiment, S17A failed significantly to reduce AWR for two out of three commercial bulb-onion cultivars, while tebuconazole reduced the final proportion of AWR-infected plants over all cultivars from 0·47 to 0·09. Combining S17A and tebuconazole resulted in a similar level of AWR to using tebuconazole alone. The use of T. viride in an integrated strategy with other treatments to enhance control of S. cepivorum is discussed.     

Evaluation of fungal antagonists for control of onion white rot in soil box trials

Four fungi ( Chaetomium globosum, Trichoderma viride, T. harzianum, Trichoderma sp.) were capable of reducing the incidence of onion white rot relative to the untreated control in two soil-box trials. When applied as a soil additive (sand: bran: fungal homogenate, 1:1:2) at the rate of 0-1% wheat bran/g dry soil, all fungal isolates provided levels of disease control equivalent to the fungicide (procymidone 0-5 g a.i./100g seed) treatment. The best results were achieved with the Chaetomium globosum and Trichoderma (C62) isolates which gave 78% and 73% control of white rot. respectively, in trial 1 and 67% and 73% control, respectively, in trial 2. Reduced control was observed when the test fungi were applied as seed coatings or incorporated into alginate pellets.     

Effect of water potential on mycelial growth and perithecial production of Monosporascus cannonballus in vitro

The effects of osmotic water potential (Ψ_s) on mycelial growth and perithecial production of Monosporascus cannonballus , the cause of root rot and vine decline of melons, were examined at 25°C on potato dextrose agar (PDA) amended with KCl, NaCl or sucrose. Patterns of the growth responses of four isolates to decreasing Ψ_s were similar for each of the osmotica. Compared with growth on nonamended PDA (−0·3 MPa), growth of all isolates increased as Ψ_s was reduced to −0·8 MPa. Maximum growth occurred at Ψ_s values of −0·6 to −0·8 MPa. Growth was not reduced below that on nonamended PDA until Ψ_s was reduced to −1·8 MPa, and a 50% reduction in growth did not occur until Ψ_s was reduced to < −2·5 MPa. Reproduction was much more sensitive to reduced Ψ_s than was mycelial growth, and perithecia were produced only at Ψ_s ≥ −0·7 or −0·8 MPa on PDA amended with KCl or NaCl, respectively. Three isolates produced perithecia on PDA amended with sucrose only at Ψ_s ≥ −0·6 MPa, but the fourth isolate produced perithecia at ≥ −1·9 MPa. Colonization of the xylem early in disease development may provide an essential source of water for subsequent reproduction in the root cortex during plant senescence. Postharvest cultivation to expose and desiccate roots may prevent reproduction even when temperatures lethal to hyphae are not attained.     

The effect of temperature and water potential on the production of conidia by sclerotia of Botrytis squamosa

The rate of conidiogenic germination of Botrytis squamosa was highest at 16°C and the greatest numbers of conidia per sclerotium (up to 5 × 10⁴) were produced at temperatures of 5–10°C. At temperatures above 20°C, the percentage of sclerotia producing conidia declined rapidly. Decreasing water potential reduced the rate at which conidia were produced and also resulted in fewer conidia produced per sclerotium. However, conidia were produced at water potentials as low as −2 MPa, at which sclerotial germination was at least 60%. A simulation model that included effects of both temperature and water potential was developed from laboratory and field data obtained for conidial production in sclerotia exposed for periods of 1, 2, 3 or 4 weeks during an entire year. There was good agreement between conidiogenic germination predicted by the model and conidial production observed in onion plots artificially inoculated with sclerotia. Temperature and water potential were therefore considered to be the principal microclimatic factors affecting conidial production by B. squamosa. The role of sclerotia in the context of UK onion production is discussed.     

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1?kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R2?=?0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.     

Effects of cultivation, conditioning and isolate on sclerotium germination in Sclerotium cepivorum

Sclerotium germination in various isolates of S. cepivorum was studied following different cultivation and conditioning treatments. A simple and rapid laboratory test was developed to trigger sclerotial germination under unsterile conditions. In most isolates, sclerotia produced under sterile and unsterile conditions showed a constitutive dormancy immediately after maturing. In contrast to this, the sclerotia of some isolates germinated quite well as soon as they were mature. The dormancy of sclerotia from axenic cultures broke down after storage in soil for 12 weeks. In a few cases sclerotia produced under unsterile conditions germinated remarkably well. Freezing and thawing of sclerotia decreased the ability of most isolates to germinate.     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: control of sclerotinia disease in glasshouse lettuce

The effects of Coniothyrium minitans inoculum quality and an 8-week interval between inoculum application and crop planting on sclerotinia ( Sclerotinia sclerotiorum ) disease in three successive lettuce crops were investigated in a glasshouse trial. Spore suspensions of three isolates of C. minitans (Conio, IVT1 and Contans) applied at 10⁸ CFU m⁻² and a standard Conio maizemeal–perlite application (06 L m⁻², 10¹¹ CFU m⁻²) were assessed for their ability to control S. sclerotiorum . Only the maizemeal–perlite inoculum (isolate Conio) consistently reduced sclerotinia disease. In the third lettuce crop only, isolates IVT1 and Contans formulated by Prophyta and isolate IVT as an oil–water formulation, all applied as spore suspensions, reduced disease at harvest compared with the untreated control. Recovery, viability and C. minitans infection of sclerotia buried during the 8-week period prior to each of the three lettuce crops, and of sclerotia formed on the crop, were tested. Only the maizemeal–perlite inoculum (isolate Conio) reduced the recovery of sclerotia buried in soil for weeks between inoculum application and crop planting, reducing their viability and increasing infection by C. minitans . Eight weeks was sufficient to enable C. minitans to infect sclerotia of S. sclerotiorum , and may account for disease control. After harvest of the second and third crops, maizemeal–perlite treatment (isolate Conio) reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore-suspension treatments. The effect of inoculum concentration and the influence of soil temperature (varying with time of year) on infection of sclerotia by C. minitans are discussed.     

Biological and integrated control of Fusarium basal rot of Narcissus using Minimedusa polyspora and other micro-organisms

In laboratory experiments several soil- and bulb-borne micro-organisms exhibited antagonism towards Fusarium oxysporum f.sp. narcissi. One-third of these micro-organisms were also found to be tolerant to thiabendazole, a fungicide commonly used for the control of this pathogen. Micro-organisms showing high levels of in vitro antagonism and/or tolerance to thiabendazole were screened in pots of natural soil artificially infested with the pathogen. Six of the micro-organisms were found significantly to suppress basal rot development when used as pre-planting bulb treatments. Minimedusa polyspora and a Streptomyces species were most effective, giving disease levels 55.72% and 56.88% lower than the control.
In a 1-year field trial pre-planting bulb treatments with M polyspora , a Streptomyces species and an isolate of Trichoderma viride significantly suppressed reductions in flower numbers, reductions in bulb yield, and percentage infection. The integration of T. viride with thiabendazole gave bulb yields significantly greater than either T. viride or thiabendazole alone whilst the integration of M. polyspora and thiabendazole resulted in significantly greater flower numbers and significantly fewer bulb infections than either treatment alone.     

Susceptibility of cultivars of garlic to Sclerotium cepivorum

Five garlic cultivars, including one claimed to be resistant to Allium white rot, were similar in their effects on the germination of sclerotia of Sclerotium cepivorum. There was no evidence of resistance to the pathogen in any of the cultivars when tested under field conditions.     

Control of bottom rot disease of lettuce (Rhizoctonia solani) using preparations of Trichoderma viride, T. harzianum or tolclofos-methyl

Isolates of Trichoderma were tested for their ability to control Rhizoctonia solani in lettuce seedlings and mature plants in a glasshouse. The best isolate (T. viride IMI 298375) was tested for its ability to control bottom rot disease in mature lettuce plants grown in polythene tunnels. Control was achieved in several trials but there was little evidence for much improvement after repeated applications and control with Trichoderma was poorer than that obtained with a standard treatment with tolclofos-methyl. Evidence was obtained to suggest however that enhanced growth of lettuce plants following treatment with Trichoderma can produce marketable yields of lettuce similar to those obtained with tolclofos-methyl.     

Differential stimulation of germination of sclerotia of Sclerotium cepivorum by cultivars of onion and its effect on white rot disease

Differences were found in the ability of onion cultivars to stimulate the germination of sclerotia of Sclerotium cepivorum under UK conditions. The differences between European and Egyptian cultivars were particularly marked. Differences also occurred in the development of white rot disease in European and Egyptian cultivars. These differences were related to the pattern of growth of the cultivars and not to levels or types of flavour and odour compounds. The effect of the pattern of growth of plants on germination of sclerotia was confirmed in experiments with different types of planting material. Germination beneath plants grown from sets was particularly rapid, but very slow beneath plants produced by direct seeding. An experiment with plants grown under different levels of sulphate nutrition showed that low levels of flavour and odour compounds were associated with a weak capacity to stimulate the germination of sclerotia. No evidence was found to suggest that control of white rot disease by selection for growth features or for differences in flavour and odour characteristics would be a practicable proposition for the foreseeable future.     

Effect of Gliocladium and Trichoderma on damping-off and blight of snapbean caused by Sclerotium rolfsii in the greenhouse

Aqueous suspensions of conidia of 285 wild-type strains and mutants of Gliocladium virens, Trichoderma hamatum, T. harzianum and T. viride were tested against Sclerotium rolfsii in the greenhouse. Ten strains of G. virens and four strains of T. harzianum suppressed damping-off of snapbean by 30-50% and blight by 36-74%. All strains of T. hamatum and T. viride tested as conidia were ineffective. In general, strains of G. virens were more effective in suppressing disease in the greenhouse than strains of T. harzianum. Several strains of G. virens and T. harzianum used alone were equal to or more effective than double and triple mixtures of such strains in disease suppression. Of four formulations of G. virens tested, germlings, alginate-bran-fermenter biomass pellets, and Pyrax-fermenter biomass mixtures reduced disease considerably and all three formulations were more effective than conidia in aqueous suspension. Strain G1-3 of G. virens added to soil as Pyrax-fermenter biomass mixtures in amounts to provide colony-forming units ranging from 1 -5 × 10³ to 1 -2 × 10⁴ per g soil provided statistically significant protection of the host at all concentrations. The extent of biological control with strains G1-3 and G1-21 of G. virens also depended on the strain of the pathogen used. Both strains effectively suppressed disease caused by strain Sr-1 (small sclerotia) of S. rolfsii. They were partially effective against strain Sr-116 (medium size sclerotia) and ineffective against strain Sr-3 (large sclerotia). Although strains G1-3 and G1-21 colonized the sclerotia of all three strains of S. rolfsii in soil effectively, they only reduced germinability of sclerotia of strain Sr-1.     

Effect of formulation on the rhizosphere competence and biocontrol ability of Trichoderma atroviride C52

The rhizosphere competence of the biological control agent Trichoderma atroviride isolate C52 was studied on onion roots both in the glasshouse and in the field when introduced into soil in a range of formulations. Proliferation of T. atroviride in the rhizosphere was formulation-dependent. A pellet formulation maintained the fungal concentration at 10⁵ cfu per g soil, whereas solid-substrate and seed-coating formulations gave concentrations of 10⁴ and 10¹ cfu per g soil, respectively. To facilitate rhizosphere-competence studies, a UP-PCR band profile generated with primer L45 for isolate C52 was used to enable conclusive identification of T. atroviride C52 when recovered from soil. When isolate C52 was introduced into Sclerotium cepivorum -infested soil as both pellet and solid-substrate formulations, there was no statistically significant difference in the disease control between these treatments, but the pellet treatment doubled the percentage of healthy plants compared with the control treatment.     

Infection of cultivars of onion, leek, garlic and A llium fistulosum by Sclerotium cepivorum

In two field experiments on land severely infested with Sclerotium cepivorum largo differences in the incidence of white rot in four Allium species were recorded. There was inuch less disease ° Cultivars of leek than in those of onion, garlic or A. fistulosum. Differences between cultivars within species were either small or absent.     

Some effects of diallyl bisulphide on sclerotia of Sclerotium cepivorum: Possible novel control method for white rot disease of onions

Sclerotium cepivorum (the causal agent of white rot of onions) persists in soil in the form of sclerotia whose germination is triggered by volatile sulphides and thiols released into the soil from roots of the genus Allium. The effects of treatment of the soil with diallyl disulphide (DADS) on the germination of these sclerotia has been studied, because if the germination can be triggered in the absence of the host crop, control of the fungus could be achieved. Five isolates of the fungus responded similarly but marked effects of DADS concentration and of the time-of-year of application were discovered, poor results being obtained with summer applications of DADS. Promising results were obtained at other times of the year with DADS flooded on to land containing sclerotia. The effectiveness of DADS treatment was shown to be related to the rate at which the material disappeared from soil, which was itself dependent upon soil temperature.     

Field testing of honeybee-dispersed Trichoderma spp. to manage sunflower head rot (Sclerotinia sclerotiorum)

Efficacy of Trichoderma spp. to reduce sunflower head rot caused by Sclerotinia sclerotiorum was evaluated in the field. A mixture of six isolates, including Trichoderma koningii , T. aureoviride and T. longibrachiatum , was tested in five field trials at Balcarce, Argentina. Trichoderma formulation (TF) included Trichoderma conidia and viable hyphal fragments, industrial talc and milled corn kernels. Honeybees ( Apis mellifera ) were used to disperse TF for six weeks from the onset of flowering. Two days after the first TF delivery, sunflower heads were inoculated with S. sclerotiorum ascospores. When 100 g TF was taken by honeybees in a 10-h per day period, head rot incidence was significantly reduced. This approach was successful in reducing disease incidence until physiological maturity of the crop, in environments highly conducive to head-rot development.     

Resistance to Sclerotium cepivorum in Allium and other genera

Stimulatory and non-stimulatory species of Allium and four other members of the Liliaceae were susceptible to infection by germinating sclerotia of Sclerotium cepivorum in unsterile soil. Plants belonging to other families were not infected in soil even when germinating sclerotia were in contact with their roots. Under aseptic conditions S. cepivorum infected plant species not closely related to Allium , particularly when large amounts of inoculum were used. Infection was slight in the presence of germinating sclerotia. It is suggested that within the genus Allium and a few related genera resistance results from the lack of ability to stimulate germination of sclerotia. Non- Allium plants also lack this ability but in addition possess tissue resistance to S. cepivorum.     

Spread of levan-positive populations of <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>, the causal agent of olive knot,in central Italy

Mixtures of wet vegetable wastes (Brassica, carrot or onion) and dry onion waste were composted at 50 °C for 7 days. The incorporation of the raw or composted vegetable waste mixtures into sandy loam, silt and peat soils reduced the viability of sclerotia of S. cepivorum in glasshouse pot bioassays. The reduction in viability was dependent on waste type, rate of incorporation, duration of exposure and soil type. Onion waste was the most effective waste type in reducing sclerotia viability in all three soils. The Brassica and carrot wastes were as effective as the onion waste in silt soil but less effective in sandy loam and peat soil. A 50% w/w incorporation rate of the wastes gave the largest reduction in viability, with an increase in reduction over time. Composted onion waste reduced sclerotia viability under glasshouse and field conditions although the effect was smaller in the field. Composted onion waste incorporated into soil at 50% w/w reduced the incidence of Allium white rot on onion seedlings in glasshouse pot tests. Incidence and control of the disease differed with soil type. The most consistent control was achieved in peat soil whereas no control was observed in silt soil. Incorporation of the waste 2 months prior to sowing or transplanting reduced seedling emergence in sandy loam soil and growth in all three soil types. The potential for field application of composted vegetable wastes as a sustainable method for control of Allium white rot and waste disposal is discussed.