Managing Our Major Greenhouse Vegetable Diseases, Using Biological & Molecular Techniques

 

Vegetable Session Canadian Greenhouse Conference October 6, 2004

 

Raj Utkhede

Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, P.O. Box 1000, Agassiz, B.C. V0M 1A0, Canada, email: utkheder@agr.gc.ca

 

Summary

            Greenhouse 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 the appropriate environmental conditions. Greenhouse vegetable growers confronted with the challenges of managing fungal pathogens have the opportunity to utilize fungi, yeasts, and bacteria as biological control agents. A number of commercially available biological products have demonstrated significant disease reduction. The use of molecular techniques in biotechnology to identify and diagnosis of plant pathogens is becoming increasingly valuable.

 

Introduction

            Vegetable crops (tomato, cucumber, pepper, and lettuce) are produced as both fresh market and processed commodities and are grown in controlled environments, such as glasshouses or other similar structures.  There are numerous fungal diseases that attack a wide range of these vegetable crops, thereby reducing crop yield and quality. Methods for disease control have included the use of cultural practices to reduce pathogen inoculums and disease incidence, development of resistant cultivars, as well as the application of chemical fungicides to inhibit pathogen development. The use of biological control strategies has also demonstrated the potential of fungi, bacteria, and yeasts in reducing a range of fungal pathogens that cause various diseases on vegetable crops.

 

            Rapid and early diagnosis of diseases would help to control the spread of devastating diseases in the greenhouses.  Identification of pathogens from culture, tissue, soil, or air samples would also help in understanding its epidemiology in greenhouse crops.  Conventional identification of pathogens from commercial greenhouse samples, using morphological parameters and pathogenicity tests needs culturing, purification and some knowledge of taxonomy and is time-consuming. Use of molecular techniques has facilitated identification of different micro-organisms quickly, efficiently and reliably and thus has helped in disease management decisions.

 

            In this paper, some examples of recent successes in biological control of fungal diseases of greenhouse vegetable crops using fungi, yeasts, and bacteria will be discussed. In particular, the research done at the Pacific Agri-Food Research Centre of Agriculture and Agri-Food Canada in Agassiz, British Columbia, Canada was discussed. In addition, the utilization of molecular techniques for quick detection and identification of fungal pathogens for disease control will be reviewed.  The diseases to be considered in this paper for which biocontrol strategies have been described include those caused by pathogenic fungi that infect the roots/crown of greenhouse vegetable crops. These include fungi such as Pythium aphanidermatum and Fusarium oxysporum.  A group of foliar-infecting fungi of vegetable crops which cause leaf spots and blights and stem infection, also have biological control strategies developed against them. These include Botrytis cinerea (grey mould), Didymella bryoniae (gummy stem blight), and a new disease called internal fruit rot on pepper caused by Fusarium subglutinans. Many different fungal, yeast, and bacterial biological control agents have been

 

identified and evaluated for disease control potential against the above-mentioned pathogens, and some have been formulated and brought to market to provide disease control options for producers of vegetable crops.  The use of biological control agents may be particularly attractive for vegetable crops grown in glasshouses, due to the high market value of these crops and the possibility for control of environmental parameters, particularly temperature and relative humidity. These are important variables that can significantly influence the efficacy of biological control agents under greenhouse conditions.  The rationale for development of biological control agents against fungal diseases on vegetable crops was to provide an additional/alternative approach to augment/replace the use of chemical fungicides, to provide a level of disease control in the absence of crop genetic resistance, and to supplement cultural control practices to further minimize the impact of these diseases and reduce chemical residues in food. 

 

Botrytis stem canker

            Botrytis cinerea, causal agent of stem canker on tomato, is renowned for its broad host range; over 200 species can be infected, resulting in considerable economic losses. On greenhouse tomatoes, this fungus causes serious and widespread stem canker and grey mould diseases. This pathogen infects leaves, petioles, stems, and fruits of the tomato crop.  Infection of leaves and stems is characterized by large, irregular, brown lesions sometimes with masses of grey-brown spores. This fungus can exploit fallen tomato petals as food bases from which it can invade green or ripening tomato fruit, either directly or by first attacking calyces attached to the fruit.  Under favorable conditions in a commercial greenhouse sporulation on lesions can be considerable with cycles of sporulation generating a fast developing epidemic within the crop.

 

Molecular technique

            A dot blot assay, based on the internal transcribed spacer of the ribosomal region, was developed for rapid diagnosis and identification of Botrytis cinerea, causal agent of grey mould of greenhouse tomatoes (Mathur and Utkhede 2002).  In this assay, DNA from fungal cultures and diseased plant tissue were amplified by PCR using primers specific to septate fungi, fixed to nylon membranes, and hybridized with digoxigenin-d-UTP labeled oligonucleotides specific to B. cinerea. The hybridized probes were detected by chemiluminescences. Of four probes screened for reaction, probe Bot1 gave a positive reaction with all isolates of B. cinerea and with fresh or frozen grey mould diseased plant tissue from Research Centre greenhouse and commercial greenhouses.  All other greenhouse fungi tested negative with this probe Bot1. This dot blot diagnoses infection by B. cinerea in large numbers of samples rapidly and reliably and distinguishes isolates of B. cinerea from other fungi found on greenhouse crops.

 

Biological control

            Experiments were conducted to identify potential antibiotic producing biological control agents by in vitro dual culture tests and to evaluate selected biological agents for control of stem canker caused by Botrytis cinerea on tomato plants grown in yellow cedar sawdust in a research greenhouse (Utkhede et al. 2001). Lesions in curative treatments with RootShield and a strain S33 of yeast (Rhodosporidium diobovatum) were significantly shorter compared with the inoculated control. Plants treated with RootShield or S33 had significantly higher total fruit yield than the inoculated control. The treatments RootShield, SoilGard, and S33 produced significantly more total fruits than the inoculated control. The number of dead plants was significantly lower in treatments RootShield and

 

S33 compared with the other treatments and inoculated control. In another set of experiments, Prestop® and S33 applied as sprays prevented the occurrence of stem canker and increased the fruit yield of tomatoes (Utkhede and Mathur (2002). The number of dead plants was also lower with these treatments compared with the other treatments and inoculated control.

 

            Additional experiments have shown that hen egg white lysozyme (HEWL), S33, and Azoxystrobin® significantly reduced lesion lengths caused by B. cinerea on cucumber plants (Utkhede and Bogdanoff 2003). HEWL plus S33 applied together did not have any effect on this cucumber disease. Nova® controlled Botrytis stem canker but not gummy stem blight.

 

Didymella Gummy Stem Blight

            Gummy stem blight of cucumber is caused by the fungus Didymella bryoniae. The first symptom on greenhouse cucumber is lesions on stubs left after the removal of fruit, tendrils or lateral shoots. These lesions may elongate and girdle the stem, causing wilt and eventual death of the plant. It is one of the serious diseases of greenhouse-grown cucumbers in the Netherlands, where it causes fruit rot. Gummy stem blight is also a problem in other European countries, including UK and Denmark. A small to moderate degree of resistance does exist in cucumber germplasm, but no gene for resistance has yet been identified. Adequate control is hard to achieve with present fungicides because plants grow fast, have dense foliage, and are continuously being wounded by picking and trimming. This disease has become a serious problem in commercial greenhouses in Canada.  Pesticides ‘Rovral’, ‘Benlate’, ‘Dyrene’ are registered for control of this disease in Canada. However, these pesticides are not very effective in controlling this disease. This may be due to development of resistant pathogens.

 

Molecular Technique

            We have developed a dot blot technique (as described earlier) for rapid diagnosis and identification of Didymella bryoniae, causal agent of gummy stem blight of greenhouse cucumbers (Koch and Utkhede 2002). After preliminary testing, probe D6 was selected for further study.  Probe D6 gave a positive reaction with all isolates of D. bryoniae and Phoma cucubitacearum tested and with fresh or frozen gummy stem blight diseased plant tissue.   All other greenhouse pathogens tested gave a negative result. Cucumber stem tissue infected with Botrytis cinerea and healthy cucumber stem tissue also gave a negative result.

 

Biological Control

            Five experiments were conducted to test chemical and biological treatments to control gummy stem blight of cucumber caused by Didymella bryoniae (Utkhede and Koch 2002). The chemical treatments ‘Nova’ and Azoxystrobin controlled the disease in 3 experiments and kresoxim-methyl in 2 experiments and the biological treatments with Enterobacter agglomerans (B8Fr) and Bacillus subtilis (AGS-4) in one experiment when applied as sprays on lesions caused by D. bryoniae on cucumbers.

 

            Another four experiments demonstrated that the treatments BASF 516, Azoxystrobin, and  Prestop® (biological agent) controlled the disease when applied as a preventative sprays on gummy stem blight lesions on cucumbers in three experiments (Utkhede and Koch 2004). Treatments with S33, Quadra 137 (Bacillus subtilis), and Decree® reduced lesion lengths compared with the control in one experiment only. No significant differences in lesion lengths were observed between RootShield®, calcium nitrate, Quadra 136 (Bacillus subtilis),  SoilGard®, Mycostop®, and inoculated control in three experiments. Azoxystrobin, BASF 516, and Prestop® had significantly less percentage of plants with gummy stem blight infection than treatments with yeast, Quadra 137, Quadra 136, RootShield®, SoilGard®, Mycostop®, Decree®, calcium nitrate, and the control.

 

In another set of experiments, we observed that lysozyme, S33, and Azoxystrobin® significantly reduced lesion lengths caused by D. bryoniae on cucumber plants (Utkhede and Bogdanoff 2003).

 

Fusarium internal fruit rot of pepper        

            Sweet pepper, grown hydroponically in greenhouses, is an important crop in Canada and around the world. About 40% of orange pepper fruits of the cultivar ‘Sympathy’ were observed to be infected with Fusarium fruit rot in a commercial BC greenhouse during 2001, and 10% in 2002 (Utkhede and Mathur 2003).  In 2003, the disease was observed on the cultivars ‘444' and ‘Spirit’ in a few commercial greenhouses in BC and Alberta. The disease appeared on mature fruits at harvest time and affected fruits are considered as culls. Infected fruits were unmarketable due to external symptoms and internal rot, resulting in losses to commercial growers. The disease appeared as discolored soft patches or necrotic spots mostly at the calyx end and some times anywhere on the mature fruit at harvest time. Seeds and surrounding area inside the fruits were covered with fungal growth and orange pink spore masses. Fungal isolations were made from the lesions. The fungus was identified as Fusarium subglutinans by Dr. Keith Seifert of Eastern Cereal and Oilseed Research Centre, Ottawa, Canada. To confirm pathogenicity, flowers and developing fruits of sweet pepper cv. ‘Sympathy’ were inoculated with F. subglutinans. About 85% of inoculated fruits and flowers developed symptoms on fruits similar to naturally infected fruits at maturity. Fruits from control plants did not develop any disease. On agar media, F. subglutinans was recovered from all inoculated infected fruits.  Preliminary study showed that this pathogen does not infect other greenhouse crops like tomatoes, cucumbers or lettuce. To our knowledge, this is the first record of fruit rot caused by F. subglutinans on greenhouse sweet peppers (Utkhede and Mathur 2003).

           

            Additional experiments were conducted to elucidate the mode of pathogen transmission and to determine the effect of inoculum’s concentration, growth stage, and various pepper cultivars on disease development (Utkhede and Mathur 2004). Inoculum concentrations of 104 to106 conidia/mL resulted in a higher incidence of fruit infection compared to 103 when just-opened and fully-opened flowers were inoculated. Flowers inoculated with F. subglutinans at different stages developed more disease as compared to fruit inoculations. None of the seeds from infected pepper fruits germinated and 92% yielded typical F. subglutinans colonies. No evidence of root infection of pepper plants by this pathogen was observed. Fruits of ‘Bison’ and ‘Mazurka’ were less susceptible to this disease than ‘444' and ‘Sympathy’.

 

Molecular Technique

            Rapid and accurate detection of F. subglutinans using dot blot hybridization was developed (Mathur and Utkhede 2004). Out of 6 probes tested, 2 probes (Fsub-3 and Fsub-5) were selected because they differentiated F. subglutinans from other Fusarium species and greenhouse pathogens. These probes can discriminate F. subglutinans from other Fusarium sp. as well as other fungal pathogens causing fruit rot of peppers. This technique would help to detect and identify accurately F. subglutinans in culture and pepper fruits.

Biological Control

            An experiment was conducted in 2003 to evaluate biologicals and chemicals as preventive treatments for control of internal fruit rot of peppers caused by Fusarium subglutinans. The inoculums of F. subglutinans was drop inoculated on flowers of sweet peppers cv. ‘Sympathy’ one day after applications of chemical and biological treatments on five different monthly dates. Pepper fruits in PreStop treatment were significantly less infected than those observed in inoculated control treatment for 4 inoculations dates. Treatments with Rovral®, BASF-516, and Q-137 showed significantly less infected fruits compared with the control on 3 inoculations dates. Pepper fruit weights were significantly higher for all biological treatments compared with the inoculated control treatment.

 

Pythium root rot on cucumber

            Cucumber plants are grown in soilless media, such as rock wool, peat, nutrient film or in sawdust, a bi-product from sawmills in British Columbia (BC). These media provide an ideal environment for dissemination and establishment of root pathogens such as Pythium species. Crown and root rot of greenhouse cucumbers in BC is caused mainly by P. aphanidermatum. Pythium crown and root rot is characterized by a rot of the basal stem and crown of the plant, accompanied by wilting and reduced growth of cucumber plants. P. aphanidermatum appears to be the most important Pythium species in this disease complex.  This fungus can spread rapidly among cucumber plants grown in a recirculating nutrient film culture system; extremely low inoculum levels of 22 colony forming units of the fungus in 100 L of nutrient solution can cause yield losses. Fungicides that are registered for control of Pythium crown and root rot of greenhouse cucumber consists of drenches of  captan and thiram at planting time, and seed treatment but are not effective. If infection is severe later in the season, growers have to replant. Since losses due to Pythium crown and root rot can approach 25-30%, there is a considerable incentive to develop alternative disease control strategies.

 

Biological control

            The potential of rhizobacterial strains to reduce the adverse effects of  Pythium root rot on plant growth and yield was examined (Utkhede et al. 1999). Of the 16 bacterial strains evaluated for antagonistic activity against this fungus, only one strain (BACT-O) of B. subtilis increased shoot growth by 9% and weight of cucumber plants by 29% compared with the inoculated controls. The same strain increased the fruit yield by 14% and fruit number by 50% of cucumber plants compared with the inoculated controls.

 

            The influence of lysozyme on Pythium root rot in cucumber was studied (Utkhede and Bogcanoff 2003). Lysozyme treatments applied as a root drench at 5 g/l every three weeks gave significantly (55%) higher yield and reduced percent infection compared with the untreated control plants.  Pythium root rot pathogen inoculation significantly reduced fresh weight and the height of cucumber seedlings.

 

Pythium root rot on lettuce

            Root diseases, caused by Pythium spp., are a particularly acute problem in re-circulating nutrient systems because these systems offer an ideal environment for root pathogens to infect and spread. Pythium spp. have been demonstrated as the most common and destructive root-infecting pathogens of lettuce in re-circulating hydroponics systems. Once these pathogens are introduced in the

 

system, their control is very difficult and sometimes the grower has to shutdown the entire system. Tiny feeder roots of lettuce produced in hydroponics systems can be infected by Pythium spp. without showing signs of obvious infection. This loss of roots can result in yield reduction. Pythium aphanidermatum, the predominant species on cucumber, can spread rapidly by means of zoospores and has been reported to be extremely virulent under hydroponics conditions, potentially destroying a crop within days.

 

Molecular technique and Biological control

            Experiments were conducted to isolate and monitor populations of Pythium spp. in re-circulating nutrient solutions at Fable Farms, Pitt meadow, and Western Lettuce Now, Langley, and to evaluate twelve chemical and biological agents to control root rot of lettuce caused by Pythium aphanidermatum (Utkhede et al. 2000). The reverse dot blot technique was used for identification of P. aphanidermatum isolated from roots of lettuce and from the nutrient solutions. The population of P. aphanidermatum in nutrient storage tanks at both locations was fairly consistent ranging from 1 to 13 per 100 ml from March 11 to July 21, 1998.

 

In experiment I, Promo and Boost have significantly increased fresh and dry lettuce weight compared with the inoculated control. The application of silicon significantly increased dry lettuce weight compared with the control. The treatments with Agrol 90, Aliette, and Ridomil were very phytotoxic to the lettuce crop at the rate used in this experiment. In experiment II, the Boost treatment at two rates, Promo at low rate, and Agrol (Amway) significantly increased fresh and dry lettuce weight compared with the control. These treatments also significantly reduced the disease ratings compared with the control.

 

Fusarium root rot on cucumber

            Fusarium root and stem rot, caused by Fusarium oxysporum f. sp. radicis- cucumerinum (FORC), is a new disease on greenhouse cucumbers that affects both yield and quality, and can result in plant death. At present, there are no control methods available for this disease. The use of fungicides in a greenhouse environment can interfere with insect biological control programs and at present, there are no effective fungicides registered for use on cucumber plants grown in soilless culture.

 

Biological control

            Three composts Cors, Dairy, and Vermi made from dairy and swine manures were tested for suppression of root and crown rot, and for the enhancement of seedling growth and fruit yield of cucumber (Kannangara and Utkhede 2001). Several bacterial colonies isolated from the composts were antagonistic to root rot pathogen and significantly increased the dry weight of four week old seedlings. The effect of compost on fruit yield was determined using compost amended sawdust, with and without inoculation of root rots pathogen. In un-inoculated plants, composts Cors, Dairy, and Vermi increased the cucumber fruit yield by 14, 17, and 23 %, respectively. In the inoculated plants yield was reduced by 13% compared to the unamended sawdust. The biggest increase (33%) in fruit yield was observed irrespective of FORC inoculations when the Dairy compost amended sawdust was inoculated with a biological agent Bacillus subtilis strain BACT-0.

 

 

Conclusions

            The potential use of microbial biological control agents for control of fungal diseases on greenhouse vegetable crops is reviewed. These microbials are easy to grow and can be applied as a seed treatment, addition to nutrient solutions or as a foliar spray. Some of the microbials appear to protect plants against a wide range of pathogens and the potential for commercial utilization is promising. The biological control agents are generally most effective when applied as a preventative treatment, prior to or at the onset of disease, and multiple applications may be needed to provide longer-term disease suppression.  At high levels of disease pressure, biological control agents can be anticipated to perform less well.  Some of the agents may be used in combination with, or in alternation with, chemical fungicides if it can be demonstrated that their survival is not adversely affected.  Similarly, it may be possible that combinations of biocontrol agents may be more effective than single organisms although little research has been done in this area.  Biological control agents that affect more than one disease should have greater market potential than those that specifically target a particular disease. One such example is that of PreStop, which has the potential to control Botrytis stem canker, Didymella gummy stem blight, Pythium root rot, and Fusarium fruit rot. The use of microbial biological control agents has generated significant interest in scientific research and product development to ensure that commercially viable products will continue to be brought to market.

 

            Correct identification of fungi by conventional means typically requires several days. Molecular methods of identification have been developed which reduce time required for a definite result to a day or two.  Molecular methods using dot blot technique would allow rapid diagnosis of disease in infected tissue from growers’ greenhouses, and would facilitate breeding resistance against plant diseases.  Rapid, accurate disease identification allows for timely disease control, and for rapid assays of propagating materials when developing disease resistant plants. In addition, the dot blot assay could be valuable for epidemiological and etiological studies, e.g. to monitor mycelial growth through the infected plant.

 

Acknowledgements

 

            Funding for some aspects of this research was provided by the British Columbia Greenhouse Growers' Association, the Canadian Inovatech Inc., the National Research Council of Canada IRAP Program, and Agriculture and Agri-Food Canada Matching Investments Initiative Program.

 

References

 

Kannangara, T. and Utkhede, R. S. (2001). Increase in fruit yield of greenhouse grown cucumber plants inoculated with Fusarium oxysporum f. sp. radicis cucumerinum by composted animal waste. Can. J. Plant Pathol. 23: 199 (abstr.)

Koch, C. A. and Utkhede, R. (2002). Diagnosis and identification of Didymellla bryoniae, causal agent of gummy stem blight of greenhouse cucumbers, using a dot blot technique. J. Horticultural Science & Biotechnology 77: 62-66.

Mathur, S. and Utkhede, R. (2002). Development of a dot blot technique for rapid identification of Botrytis cinerea, the causal organism for grey mould in greenhouse tomatoes.  J. Horticultural Science & Biotechnology 77: 604-608.

Mathur, S. and Utkhede, R. (2004). Dot blot hybridization technique for identification and diagnosis of Fusarium subglutinans fruit rot in greenhouse peppers. Can. J. Plant Pathol. 26: 123 (abstr.).

Utkhede, R. S., Koch, C.A., and Menzies, J.G. (1999). Rhizobacterial growth and yield promotion of cucumber plants inoculated with Pythium aphanidermatum. Can. J. Plant Pathol. 21: 265- 271.

Utkhede, R. S., Levesque, C. A., and Dinh, D. (2000). Pythium aphanidermatum root rot in hydroponically grown lettuce and the effect of chemical and biological agents on its control. Can. J. Plant Pathol. 22: 138-144.

Utkhede, R., Bogdanoff, C. and McNevin, J. (2001). Effects of biological and chemical treatments on Botrytis stem canker and fruit yield of tomato under greenhouse conditions. Can. J. Plant Pathol. 23: 253-259.

Utkhede, R. and Mathur, S. (2002). Biological control of stem canker of greenhouse tomatoes caused by Botrytis cinerea. Can. J. Microbiol. 48: 550-554.

Utkhede, R. S. and Koch, C. A. (2002). Chemical and biological treatments for control of gummy stem blight of greenhouse cucumbers. European J. Plant Pathol. 108: 443-448.

Utkhede, R. and Bogdnoff, C. (2003).  Influence of lysozyme, yeast, Azoxystrobin®, and Nova® on fungal diseases of cucumbers grown hydroponically. Crop Protection 22: 315-320.

Utkhede, R, and Mathur, S. (2003). Fusarium fruit rot of greenhouse peppers in Canada. Plant Dis.  87:100 (Abstr.).

Utkhede, R. S. and Koch, C. A. (2004). Evaluation of biological and chemical treatments for control of gummy stem blight on cucumber plants grown hydroponically in greenhouses. BioControl 49: 109-117.

Utkhede, R, and Mathur, S. (2004). Internal fruit rot of greenhouse grown sweet peppers caused by Fusarium subglutinans. Can. J. Plant Pathol. (In press).