R.S. Utkhede

Using fungi and yeasts to manage vegetable crop diseases

Vegetable crops are grown worldwide as a source of nutrients and fiber in the human diet. Fungal plant pathogens can cause devastation in these crops under appropriate environmental conditions. Vegetable producers confronted with the challenges of managing fungal pathogens have the opportunity to use fungi and yeasts as biological control agents. Several commercially available products have shown significant disease reduction through various mechanisms to reduce pathogen development and disease. Production of hydrolytic enzymes and antibiotics, competition for plant nutrients and niche colonization, induction of plant host defense mechanisms, and interference with pathogenicity factors in the pathogen are the most important mechanisms. Biotechnological techniques are becoming increasingly valuable to elucidate the mechanisms of action of fungi and yeasts and provide genetic characterization and molecular markers to monitor the spread of these agents.

Evaluation of plant growth-promoting rhizobacteria for biological control of pythium root rot of cucumbers grown in rockwool and effects on yield

Three strains ofPseudomonas fluorescens (63-49, 63-28, and 15), one strain ofPseudomonas corrugata (13) and one strain ofSerratia plymuthica (R1GC4) were tested on rockwool-grown cucumbers for their ability to reduce Pythium root-rot caused byPythium aphanidermatum. These strains were previously selected for biocontrol ability from collections of >4000 bacteria. Strains 63-49 and 63-28 were tested on cucumber plants grown in rockwool in two replicatedPythium-inoculated trials conducted in British Columbia (B.C). Another inoculated, replicated trial was conducted in Quebec with all five strains. Cucumber yields (fruit number and weight) were measured over a ten-week harvest period. Strain 63-49 caused an early promotion of plant growth and increased cucumber yields at early harvests. No measurable effect ofPythium inoculation on disease development was observed in the Quebec trial, due to unfavourable cool weather. However, 63-49 significantly increased the total number of cucumbers (12%) and cucumber weight (18%), compared to the non-treated control. Strains 13, 15 and R1GC4 slightly increased the cumulative cucumber yields, but strain 63-28 had no effect. In the B.C. trial, inoculation withP. aphanidermatum reduced the number and weight of cucumbers by 27%. Treatments ofPythium-inoculated cucumbers with 63-49 significantly increased fruit number and weight by 18%, compared to thePythium-inoculated control. Strain 63-28 increased the cumulative number of cucumbers over time, compared to thePythium-inoculated control, but the increase was less than with 63-49. The use ofPseudomonas spp. in rockwool-grown cucumbers can increase yields, both in the presence and absence of Pythium root rot, and with variable seasonal conditions and disease pressures.

Biological treatments to control bacterial canker of greenhouse tomatoes

Experiments were conducted to determine the effects of treatments on Clavibacter michiganensis subsp. michiganensis in vitro and on young seedlingsinoculated with the pathogen under greenhouseconditions. Lysozyme was bactericidal at 10 g/l concentration in vitro. Tomato plantstreated with lysozyme at 10 g/l and 100 g/lshowed significantly higher plant heightcompared with the inoculated control plants,and plants in these treatments were as tall asthose observed in untreated uninoculatedcontrol plants. Treatments with B. subtilis (Quadra 136) and Trichoderma harzianum (RootShield®), lysozyme,vermicompostea, Rhodosporidium diobovatum(S33), B. subtilis (Quadra 137) appliedas a spray at 0.3 g/l, 0.6 g/l, 10 g/l,concentrated, 1 × 109 CFU/ml, and 0.5 g/l,respectively, have the ability to prevent theincidence of bacterial canker of tomato plantscaused by C. michiganensis subsp.michiganensis under greenhouse conditions.

Increased Growth and Yield of Hydroponically Grown Greenhouse Tomato Plants Inoculated with Arbuscular Mycorrhizal Fungi and Fusarium oxysporum f. sp. radicis-lycopersici

The arbuscular mycorrhizal fungi Glomus monosporum, G. vesiculiferum, G. deserticola, G. intraradices, G. mosseae, and two unidentified species were tested to determine their effect on plant growth and fruit production of tomato (Lycopersicon esculentum Mill.) cv. Trust inoculated with Fusarium oxysporum f. sp. radicis-lycopersici (FORL) under near-commercial greenhouse conditions. Inoculation with G. monosporum and G. mosseae significantly increased fruit yield and fruit number of tomato plants grown hydroponically in sawdust. Plant height and plant dry weight increased significantly when inoculated with G. monosporum and G. mosseae. Further, plants inoculated with G. monosporum and G. mosseae showed significantly lower FORL root infection than the untreated control plants.

Foliar applications of fertilizer salts inhibit powdery mildew on tomato

Foliar applications of a number of inorganic fertilizer salts were found to significantly reduce powdery mildew [Erysiphe orontii] on greenhouse tomato (Lycopersicon esculentum, cv. Trust) leaves. In a series of single-application experiments, the foliar applications, each with 0.1% surfactant, were applied to the third and fourth leaves of young tomato plants 24 h before inoculation with an atomized application of mildew conidia. Control treatments consisted of a water application and a water plus surfactant application. Powdery mildew colonies were counted 7–10 days later. Surfactant alone significantly reduced mildew colony numbers. CaCl2, Ca(NO3)2, and K2HPO4 reduced colony counts compared with the surfactant alone. All combinations of Ca salts, or Ca salts plus elemental S, significantly reduced mildew colony counts compared with surfactant alone. In a second set of experiments, the effects of repeated applications on already naturally infected tomato plants were evaluated. Young tomato plants were moved to a greenhouse containing several heavily mildew-infected tomato plants, allowing natural infection. The following day and every 7–10 days thereafter, treatments were applied to all leaves. Colony counts on selected leaves were made weekly or just before spraying. Surfactant alone was not as effective as in the single-application treatments, often having no effect. All the Ca-salt treatments that were effective in the single-application series were effective as multiple applications. Repeated applications of combinations of Ca salts were often just as effective as applications of elemental S. KCl, MgSO4, and K2HPO4 also significantly reduced mildew counts with multiple applications. This study did not attempt to explain the differences or similarities in efficacy of the salts tested; both osmotic (concentration) and specific-ion effects could play a role.Key words: surfactant, Erysiphe orontii, Lycopersicon esculentum, greenhouse, calcium, potassium, sulphur, magnesium, powdery mildew.

Preventive and Curative Biological Treatments for Control of Botrytis cinerea Stem Canker of Greenhouse Tomatoes

The effects of biological treatments with PlantShield®, Prestop®, Quadra 136, RootShield®, and S33 (Rhodosporidium diobovatum) and chemical treatment with Decree® applied as a preventive or curative sprays on stem canker caused by Botrytis cinerea on tomato plants grown in sawdust were studied under near-commercial greenhouse conditions. Prestop® and Decree®, applied as preventive or curative sprays, PlantShield® applied as curative spray, and S33 and Q-136 applied as preventive or preventive plus one spray to wounded surface provided season-long protection from B. cinerea stem canker. These treatments also increased fruit yield and decreased the number of dead plants compared with the inoculated control.

Chemical and biological treatments for control of gummy stem blight of greenhouse cucumbers.

Experiments were conducted to determine the effects of chemical and biological treatments on gummy stem blight of cucumber caused by Didymella bryoniae in vitro and under greenhouse conditions. Eleven strains of Bacillus subtilis, strain AGB10 of B. cereus, and strain B8Fr of Enterobacter agglomerans produced antagonistic zone against D. bryoniae in vitro. Of four experiments conducted, the chemical treatments ‘Nova’,1 kresoxim-methyl, and azoxystrobin controlled the disease in three experiments and the biological treatments E. agglomerans (B8Fr), B. subtilis (AGS-4), and lysozyme in one experiment when applied as sprays on lesions caused by D. bryoniae on cucumber plants under greenhouse conditions. Fruit rot of cucumber was significantly reduced when the fruit was treated with ‘Nova’ or kresoxim-methyl. These results suggest the potential of azoxystrobin, kresoxim-methyl, E. agglomerans (B8Fr), and B. subtilis (AGS-4) applied post-inoculation to control gummy stem blight on greenhouse cucumbers.

Development of a multiplex classical polymerase chain reaction technique for detection of Didymella bryoniae in infected cucumber tissues and greenhouse air samples

Two sets of primer pairs were evaluated for their usefulness in detecting and identifying Didymella bryoniae (anamorph Phoma cucurbitacearum), causal agent of gummy stem blight, in infected cucumber plant tissues and air samples, using polymerase chain reaction. One primer in each pair was a universal primer that has been used for amplification of septate fungi. The other primer in each pair was an oligonucleotide designed from sequence information in the GenBank database to be specific to D. bryoniae. Each primer pair on its own was specific to genus level, but gave some nonspecific results for other Phoma or Didymella species. However, when these primer pairs were combined in a multiplex PCR, a unique result for D. bryoniae was obtained. The multiplex PCR gave positive results for cucumber tissue infected with D. bryoniae, and negative results for uninfected tissue and for tissue infected with Botrytis cinerea. Positive results were obtained from air samples collected with a rotation impaction sampler situated in a greenhouse containing cucumber plants infected with gummy stem blight. This method is rapid, sensitive, and accurate for detecting and identifying D. bryoniae in pure culture and plant tissue and could be applied to the detection of this organism in air samples. This method, therefore, could be a valuable technique for use in disease prediction.

Internal fruit rot caused by Fusarium subglutinans in greenhouse sweet peppers

Experiments were conducted to isolate the causal organism of internal fruit rot in greenhouse sweet pepper (Capsicum annum), confirm its identity, and elucidate its transmission mode and host range. The effects of inoculum concentration, growth stage of the plant, and cultivar of sweet pepper on disease development were also determined. The causal organism was identified as Fusarium subglutinans. Inoculum concentrations of 104 to 106 conidia/mL resulted in a higher incidence of fruit infection compared with 103 conidia/mL when just-opened and fully opened flowers were inoculated. Inoculation of flowers at different stages with F. subglutinans resulted in more diseased fruits than fruit inoculations. None of the seeds from infected pepper fruits germinated, and 92% of those yielded typical F. subglutinans colonies. No evidence of root infection of pepper plants by F. subglutinans was observed, and no disease developed on fruits of greenhouse tomato, cucumber, or eggplant. Fruits of Capsicum annuum ‘Bison’ and ‘Mazurka’ were less susceptible to F. subglutinans than those of ‘Sympathy’ and ‘444’.

Molecular detection of Fusarium subglutinans, the causal organism of internal fruit rot in greenhouse peppers

Internal fruit rot of sweet peppers, caused by Fusarium subglutinans is a new disease found in commercial greenhouses in British Columbia and Alberta, which causes considerable yield losses. Experiments were conducted to develop a rapid and accurate assay for detection of F. subglutinans, by dot-blot hybridization. Internal transcribed spacers 1 (ITS1) and 2 (ITS2) and 5.8S rDNA were amplified by polymerase chain reaction, using universal primers, and a dot-blot assay was employed to detect the pathogen in culture and in the host. Among six probes tested, three (Fsub-1, Fsub-3, and Fsub-5) were selected because they differentiated F. subglutinans from other Fusarium spp. and greenhouse pathogens. Probe Fsub-3 hybridized with all F. subglutinans isolates. Fsub-1 hybridized only with F. subglutinans isolates from Monterey pine (Pinus radiata). Fsub-5 hybridized with F. subglutinans isolates from corn (Zea mays), peppers (Capsicum annuum), and Panicum miliaceum, but not with Pinus radiata isolates. None of these primers hybridized with Fusarium spp. other than F. subglutinans or with other pathogens of greenhouse crops. Dot-blot hybridization developed in this study differentiates F. subglutinans from other Fusarium spp. as well as from other fungal pathogens causing fruit rot of peppers. This technique may help to detect and identify F. subglutinans in culture and in pepper fruits.