Control of Pythium damping-off of sugar beet by seed treatment with crop straw powders and a biocontrol agent

Seed treatment with non-sterilized powdered straws from 39 crops was tested for the control of Pythium damping-off of sugar beet. Four straws, including flax, coriander, pea, and lentil were effective in controlling the disease in soil artificially infested with Pythium sp. “group G.” Sterilizing flax and pea straws eliminated the efficacy of these straws. Wheat straw powder coated on sugar beet seeds increased the incidence of Pythium damping-off but this effect was reversed by the co-inoculation of wheat straws with the biocontrol agent Pseudomonas fluorescens 708. Coating sugar beet seeds with P. fluorescens 708 and flax or pea straws also increased the efficiency of the bacterial strain for the control of Pythium damping-off. Pea straws and to a lesser extent lentil straws produced volatile substances that affected mycelial growth of Pythium sp. “group G” on potato dextrose agar in Petri plates when the straws were mixed with water and left to ferment for two days. Fermentation of pea straws led to the accumulation of volatile ammonia, which was produced by the reduction of the large amount of nitrate stored in the straw. Reduction of nitrate and therefore the release of volatile ammonia did not occur in sterilized pea straws. However, fermenting sterile pea straws with bacteria from different genera restored nitrate reduction and the release of volatile ammonia, suggesting that microorganisms associated with pea straws are responsible for the conversion of nitrate into volatile ammonia which in turn control Pythium damping-off disease in sugar beet.     

Possible Role of Xanthobaccins Produced by Stenotrophomonas sp. Strain SB-K88 in Suppression of Sugar Beet Damping-Off Disease

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 μg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

The influence ofPythium on the growth of barley seedlings as affected by soil water and inoculum density

Summary Pythium spp. were more abundant in the southerly and more temperature regions of the barley growing region of South Australia than in the drier and hotter north. Populations were more abundant in the top 10 cm than in the 10 to 20 cm soil zone. Eleven species ofPythium were identified from barley crops.P. irregulare appeared to be the most abundant and was one of the most pathogenic species on barley.P. volutum was also highly pathogenic; it had not been recorded in South Australia before. A factorial experiment using ninePythium spp. and four levels of soil water indicated that unlike other species,P. irregulare, P. volutum andP. graminicolum were most pathogenic in soils with a water content close to field capacity. A factorial experiment usingP. irregulare at four levels of soil water and six inoculum levels showed that inhibition of growth in barley seedlings byP. irregulare increased as the level of water in the soil increased. The experiments support the hypothesis that inhibition of growth of barley seedlings byPythium spp. is most severe in the southerly parts of the barley growing area of South Australia particularly where there is a combination of high soil water and high population densities.     

The effect of D–D, chloropicrin and previous crops on numbers of migratory root-parasitic nematodes and on the growth of sugar beet and barley

Thirty-three gal D–D or chloropicrin/acre (371 l/ha) injected during winter into well-drained, sandy soils controlled Longidorus attenuatus, Trichodorus spp. and other migratory root-parasitic nematodes and resulted in greatly increased yields of sugar beet for at least 3 years; 2 years of bare fallow was less effective than soil fumigation. Trichodorus spp. multiplied more on sugar beet than on barley, whereas L. attenuatus multiplied more on barley and clover than on sugar beet.     

The use of mutant and engineered microbial agents for biological control of plant diseases caused by Pythium: Achievements versus challenges

Pythium species are devasting pathogens causing major crop losses, e.g., damping-off in sugar beet caused by Pythium ultimum and root-rot of tomato caused by Pythium aphanidermatum. The use of natural antagonistic microorganisms is a promising environment-friendly approach to control Pythium-caused plant diseases. There are several examples of biocontrol of diseases caused by Pythium species but the application of bioeffectors (biological control agents) is limited for various reasons, including the restricted amount of gene-modification based biotechnological progress. The regulations in many countries prevent genetically modified bioeffectors from being routinely deployed in field conditions. Our two connected aims in this review are (1) to compile and assess achievements in genetic modification of bioeffectors which have been tested for parasitism or antagonism towards a Pythium plant pathogen or biocontrol of a plant disease caused by a Pythium species, and (2) discuss how a better performing bioeffector could be engineered to improve biocontrol of Pythium-caused plant diseases. We focus on the role of seven key mechanisms: cellulases, carbon catabolite de-repression, glycosylation, reactive oxygen species, chitin re-modelling, proteases, and toxic secondary metabolites. Genetic modifications of bioeffectors include gene deletion and overexpression, as well as the replacement of promoter elements to tune the gene expression to the presence of the pathogen. Gene-modifications are limited to fungal and bacterial bioeffectors due to the difficulty of gene modification in oomycete bioeffectors such as Pythium oligandrum. We assess how previous gene modifications could be combined and what other gene modification techniques could be introduced to make improved bioeffectors for Pythium-caused plant diseases. The broad host-range of Pythium spp. suggests engineering improved antagonistic traits of a bioeffector could be more effective than engineering plant-mediated traits i.e., engineer a bioeffector to antagonise a plant pathogen in common with multiple plant hosts rather than prime each unique plant host.     

Effects of Pythium species on the growth of apple and their possible causal role in apple replant disease

Several lines of circumstantial evidence collectively indicated that poor early growth of apple (‘replant disease’) might be associated with the effects of soil-borne pythiaceous fungi. This hypothesis was supported by pathogenicity tests. All isolates tested of P. sylvaticum and certain isolates of seven other Pythium spp. significantly reduced the growth of apple seedlings. The growth reductions caused by certain Pythium isolates were of comparable magnitude to the growth increases occurring after chloropicrin-fumigation of apple orchard soils. The Pythium isolates most virulent to apple were of low virulence to a clonal cherry rootstock. Reappraisal of the nature of the disease as a non-specific soil malaise is consistent with established features of the pathology of Pythium spp. The disease, however, is an ill-defined ‘poor growth phenomenon’ with no diagnostic symptoms and conclusive evidence that Pythium spp. are widely causal is likely to be elusive.     

Antagonistic activity of bacteria isolated from crops cultivated in a rotation system and a monoculture against <Emphasis Type="Italic">Pythium debaryanum</Emphasis> and <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The effect of crop rotation and monocropping on the occurrence of bacteria with antagonistic activity toward Pythium debaryanum and Fusarium oxysporum was shown. Arthrobacter spp., fluorescent Pseudomonas spp. and actinomycetes were isolated from winter rape, sugar beet and winter barley rhizosphere and bulk soil from the plots of a long-term crop rotation experiment (18 years). The occurrence of mycoantagonistic isolates and their antibiosis level exhibited specificity for the site, crop and crop rotation. Mycoantagonistic activity was common among actinomycetes and fluorescent Pseudomonas spp. and less frequent among Arthrobacter spp. Antibiosis of fluorescent Pseudomonas spp. and Arthrobacter spp. was in general stronger against P. debaryanum than F. oxysporum. The highest percentage of antagonistic Pseudomonas spp. against P. debaryanum was in the plots of barley crop, while plots of winter rape showed higher frequency of antagonists against F. oxysporum. The highest antibiosis activity of Arthrobacter spp. against both pathogens occurred in isolates from barley and winter rape monoculture, and there were no F. oxysporum antagonists among these bacteria in sugar beet monoculture. Most of actinomycete isolates strongly inhibited growth of P. debaryanum and F. oxysporum. The percentage of mycoantagonistic actinomycetes and their antibiosis level were the highest in the 6-year crop rotation system.     

Survival and recovery of Phytophthora capsici and oomycetes in tailwater and soil from vegetable fields in Florida

A current trend in Florida agriculture to conserve water is to irrigate with surface runoff water (tailwater) recovered in retention ponds and canals. Water filtration and lemon leaf baiting recovered Phytophthora capsici and other plant pathogenic Oomycetes in runoff water from ponds and canals. A total of 196 isolates of Phytophthora spp. and 471 isolates of Pythium spp. were recovered. Phytophthora spp. included P. capsici, P. cinnamomi, P. lateralis, P. nicotianae, P. citricola, P. cryptogea and P. erythroseptica. Species of Pythium were P. aphanidermatum, P. catenulatum, P. helicoides, P. irregulare, P. myriotylum, and Pythium‘group F’. Isolates of P. aphanidermatum, P. irregulare, P. myriotylum, and Pythium‘group F’ were pathogenic on pepper and tomato. Recovery of P. capsici propagules was related to soil moisture‐holding capacity and time interval but not temperature. Recovery of P. capsici propagules at 100% soil moisture‐holding capacity and 30° C was 57 days. In tailwater, recovery of propagules of P. capsici was 63 days at 24°C to 25°C. The potential exists to reintroduce and disseminate species of Phytophthora and Pythium when using tailwater for irrigation or other practices.     

Antagonistic Activities of Trichoderma harzianum Against Pythium aphanidermatum and Pythium myriotylum on Tobacco

Trichoderma harzianum rendered Pythium aphanidermatum and P. myriotylum non-viable in Petri dish dual culture. The Pythium mycelia from such cultures showed natural autofluorescence in the regions of interactions, indicating their death. Non-volatile and volatile fungicidal activities were detected in T. harzianum culture. Lytic activity of β-(l,3)-glucanase was detected on the cell walls of the Pythium spp. There was a significant decrease in the disease incidence when T. harzianum was incorporated into sterile soil, whereas the effect was insignificant in natural soil.     

The effect of fumigants on soil recolonization in the glasshouse by organisms colonizing the spermosphere

Germinating turnip seeds were used as baits to measure changes in colonizing floras of the spermosphere in glasshouse soil boxes following fumigation. The spermosphere mycoflora of the untreated soil was always dominated by Fusarium, Pythium, and Gliocladium spp. Methyl bromide, chloropicrin, and MBC₃₃ (a 67:33 mixture of methyl bromide and chloropicrin), applied in polyethylene bags at a rate of 2·5 ml/cubic foot of soil, greatly reduced colonization by Fusarium and Pythium spp. for 120 days. Fungi and actinomycetes recolonizing treated soils were more active than in untreated soils, and each fumigant induced a characteristic recolonization pattern. The spermosphere of methyl bromide-treated soil was initially dominated by actinomycetes and later by Penicillium spp. In chloropicrin-treated soil, Trichoderma and later Penicillium spp. dominated, and in soil treated with MBC ₃₃, Penicillium and Trichoderma spp. were co-dominant. Dilution plates, made at the same time from the same soil, indicated a close correlation between inoculum density of different fungi in the soil and their inoculum potentials towards the spermosphere.     

Attraction and oviposition responses of the fungus gnat Bradysia impatiens to microbes and microbe‐inoculated seedlings in laboratory bioassays

Laboratory tests were conducted to examine preferences of Bradysia impatiens Johannsen (Diptera: Sciaridae) larvae and adults for various microbes associated with greenhouse crops. Fungus gnat larvae and adults exhibited a preference for cultures of Pythium spp. over the medium used to grow the pathogens. Larvae also exhibited a preference for geranium seedlings infected with pathogenic Pythium spp. [P. aphanidermatum (Edson) Fitz., P. ultimum Trow, and P. irregulare Buis. (Oomycota: Peronosporales)] over non‐inoculated plants. Adult fungus gnats exhibited a strong ovipositional preference for the aforementioned Pythium spp. as well as a variety of other microorganisms, including the pathogenic fungus Thielaviopsis basicola (Berk. & Br.) (Ascomycota: Microascales), the geranium‐infecting bacterium Xanthomonas campestris pv. pelargonii (Brown) Dye (Proteobacteria: Xanthomonadales), the non‐pathogenic species Pythium torulosum Coker & P. Patt. and Pythium graminicola Subramaniam, the pathogen‐suppressive fungus Trichoderma harzianum Rifai (Ascomycota: Hypocreales), and the insect pathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales). Our study is the first to demonstrate that fungus gnats are attracted to and/or stimulated to oviposit by a wide array of living microorganisms both in pure culture and in association with plant seedlings. These findings have important implications with respect to the potential role of fungus gnats in plant pathogen transmission.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

Phytophthora and Pythium Associated with Feeder Root Rot of Citrus in the Transvaal Province of South Africa

Mild to extensive feeder root rot was present in all 23 orchards, with trees showing symptoms of citrus decline from nine areas in the Transvaal Province of South Africa. Phytophthora nicotianae and Pythium spp. were isolated from diseased roots and rhizosphere soils in all areas sampled. Isolations from diseased feeder roots showed P. nicotianae present in 26% of orchards during Spring and 61% of orchards during Autumn, while Pythium spp. were present in 56% of orchards during Spring and 65% of orchards during Autumn. In isolations from baited rhizosphere soils, P. nicotianae was present in 56% of orchards during Spring and 52% of orchards during Autumn, while Pythium spp. were present in 69% of orchards during Spring and 82% of orchards during Autumn. In rhizosphere soils, the mean population density of Pythium spp. was higher than that of P. nicotianae throughout the season. Only P. nicotianae was consistently isolated during thesurvey. Different Pythium spp. were isolated of which two were tentatively identified as P. paroecandrum and Pythium‘Gp.G’.     

Additional experiences to elucidate the microbial component of soil suppressiveness towards strawberry black root rot complex

Several studies were carried out to investigate the soil microbial components involved in suppressing strawberry black rot root which occurs throughout the Italian strawberry growing region. Quantitative and qualitative evaluation of fungi involved in black root rot were combined with several soil microbial parameters involved in soil suppressiveness towards black root rot agents. The first survey, carried out in an intensively cultivated area of northern Italy, identified Rhizoctonia spp. as the main root pathogen together with several typical weak pathogens belonging to the well‐known black rot root complex of strawberry crop: Cylindrocarpondestructans, Fusarium oxysporum, F. solani, Pestalotia longiseta and others. The root colonisation frequency of strawberry plants increased strongly from autumn to spring at harvesting stage. Rhizoctonia spp. were the only pathogens which followed the rising trend of root colonisation with relative frequency; all the weak pathogens of strawberry black root rot complex did not vary their frequency. Only non‐pathogenic fungi decreased from autumn to spring when at least 60% of colonising fungi were represented by Rhizoctonia. These data suggested that the late vegetative stage was the best time to record the soil inoculum of root rot agents in strawberry using root infection frequency as a parameter of soil health. A further study was performed in two fields, chosen for their common soil texture and pH, but with significant differences in previous soil management: one (ALSIA) had been subjected to strawberry monoculture without organic input for several years; the other (CIF) has been managed according to a 4‐year crop rotation and high organic input. In this study Pythium artificially inoculated was adopted as an indicator for the behaviour of saprophytically living pathogens in bulk soil. Pythium showed a sharp, different response after inoculation in bulk soil from the two soil systems evaluated. Pythium was suppressed only in the CIF field where the highest levels of total fungi and fluorescent bacteria and highest variability were observed. The suppressiveness conditions towards Pythium, observed in the CIF and absent in the ALSIA field, corresponded with the root infection frequency recorded at the late vegetative stage on strawberry plants grown in the two fields: strawberry plants from the CIF field showed lower root colonisation frequency and higher variability than that recorded on those coming from the ALSIA field.     

Stilbella aciculosa: A Potential Biocontrol Fungus against Rhizoctonia solani

A semi‐solid fermentation product of the potential biocontrol fungus Stilbella aciculosa was formulated on wheat bran: water (1:1, w/w) and incubated 5, 10 and 15 days before addition to soil infested with the pathogen Rhizoctonia solani. Generally, preparations did not reduce survival of the pathogen in infested beet seed but they did prevent saprophytic growth of the pathogen from beet seed into soil. The magnitude of reduction by the 15‐day‐old inoculum was greater than that by the 5‐day‐old inoculum. Ten‐day‐old bran preparations of S. aciculosa at rates of 0.5 and 1.0% (w/w) in soil prevented post‐emergence damping‐off of cotton, radish and sugar beet in the glasshouse and a rate of 1.0% gave stands similar to those in the non‐infested control soil. The antagonist, grown on perlite formulated with molasses, cornmeal, alfalfa tissue or corn stover, prevented damping‐off of cotton in a naturally infested soil. However, the stands were not as great as that in soil planted with pentachloronitrobenzene (PCNB)‐treated seed. Toxic metabolites, produced by S. aciculosa developing on various substrates, slightly inhibited the growth of R. solani in culture and induced cytoplasmic leakage of the pathogen mycelium.     

Enhanced activity of introduced biocontrol agents in solarized soils and its implications on the integrated control of tomato damping-off caused by Pythium spp

Soil solarization in combination with introduction of biocontrol agents (BCA) was evaluated as a potential disease management strategy for tomato damping-off caused by Pythium spp. A rifampicin resistant Pseudomonas fluorescens strain (PfT-8) and a carbendazim resistant Trichoderma harzianum strain (ThM-1) were introduced into soil following solarization. Tomato seeds were planted into treated field plots. The influence of soil solarization and application of biocontrol agents on damping-off incidence, plant biomass, rhizosphere population of introduced antagonists, and native Pythium spp. was assessed by two consecutive field trials. Damping-off incidence was significantly reduced in solarized plots compared to control. Soil inoculation of biocontrol agents into solarized plots resulted in the highest suppression of damping-off incidence (PfT-8 up to 92%; ThM-1 up to 83%), and increase in plant biomass (PfT-8 up to 66%; ThM-1 up to 48%) when compared to un-solarized control plots. Rhizosphere population of introduced biocontrol agents gradually increased (PfT-8 up to 102% and ThM-1 up to 84%) in solarized soils when compared to unsolarized control. The population of Pythium spp in rhizosphere soil was reduced up to 55% in solarized plots; whereas, application of BCA to solarized soils reduced the rhizosphere population of Pythium spp. by 86 and 82% in P. fluorescens and T. harzianum applied plots respectively.     

Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

Biochemical characterisation of Pythium spp. involved in cavity spot of carrots in France

The pathogenicity and growth rate in vivo were assessed on 27 isolates of Pythium spp. recovered from cavity spot lesions on carrots grown in various parts of northwest France. Polyacrylamide gel electrophoresis of isoesterases was used to identify the Pythium spp. involved. Slow-growing isolates were more aggressive than fast-growing ones when inoculated on carrot tap roots. Isoesterase patterns identified the slow-growing isolates as P. violae and P. sulcatum; P. ultimum and P. intermedium were identified among the less aggressive fast-growing isolate group, in which some isolates were also classed as P. sylvaticum or P. irregulare, which have similar electrophoretic profiles. The incidence of Pythium spp. associated with the disease in France is discussed in regard to cavity spot in other countries.     

Field assessment of soil biological and chemical quality in response to crop management practices

Soil microbiological and chemical aspects were evaluated to determine the effects of conservation tillage and crop rotation on soil fertility over a 16-year period. A field trial was established to compare two cropping systems (continuous soybean and maize/soybean, soybean/maize rotation). In addition, maize (Zea mays L.) and soybean (Glycine max L., Merr) were grown in two different tillage systems: no tillage and reduced tillage. Soil populations of Trichoderma spp., Gliocladium spp. and total fungi were more abundant when maize or soybean were under conservation tillage and in the maize/soybean and soybean/maize rotation, than in continuous soybean. Furthermore, higher levels of microbial respiration and fluorescein diacetate hydrolysis (FDA), were recorded under no tillage systems. However, soil counts of Actinomycetes and Pythium spp., and Pythium diversity together with soil microbial biomass were not affected by the field treatments. To establish a correlation with soil biological factors, soil chemical parameters, such as pH, organic matter content, total N, electrical conductivity, N–NO₃ ⁻ and P were also quantified, most of the correlations being significantly positive. Under no tillage there was a clear increase of the amount of crop residues and the C and N soil content due to the presence of residues. Also the distribution of crop residues in surface soil due to zero tillage and the quality of these residues, depending on the crop rotation employed, improved on soil biological and chemical characteristics. Crop yield was also enhanced by zero tillage through the management of residues. Although yield values were not directly associated with the development of microorganisms, both yield and microorganisms were influenced by crop management. These results suggest that measuring soil properties over a long period helps to define effective management strategies in order to preserve soil conditions.