Marina Stocco

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.

Biological control of leaf grey mould of greenhouse tomatoes caused by Botrytis cinerea

Botrytis cinerea (grey mould) is one of the most common diseases of glasshouse crops and is a major cause of plant death in long-season tomato production. A disease management field trial was conducted in a commercial tomato production greenhouse for comparison of seven fungal antagonists which had previously exhibited potential as biological control agents with respect to Botrytis cinerea incidence and severity. Isolates of Trichoderma harzianum, Candida pelliculosa, Rhodotorula rubra, and Fusarium semitectum were recovered from leaves, fruits and flowers of different solanaceous plants. The fungal antagonists were tested for their control of leaf grey mould on tomato under greenhouse conditions during 2008 and 2009. In both years, foliar spray with strains of Trichoderma harzianum and Fusarium semitectum suppressed the foliar infection of B. cinerea. The suspensions of T. harzianum 118 and 252 and F. semitectum 25 significantly reduced disease incidence (65–95%) and severity (50–77%) in inoculated plants compared to untreated controls (P ≤ 0.05). Our results suggest that formulations of saprophytic fungi selected from the naturally occurring mycoflora could be an effective tool in the biological control of tomato grey mould.

A comparison of preservation methods for Trichoderma harzianum cultures

To eliminate problems encountered by serial transfers of stock cultures, several methods for long-term preservation have been developed. Most methods slow metabolism of microorganisms by storage at 4 1C and/or mineral oil coating, thereby limiting oxygen uptake by the cell, decreasing metabolic activities, and increasing storage time. One storage method using cellulose filter paper has been little used in mycological collections. In the present study, we conducted experiments to evaluate several of these methods, including the cellulose filter paper technique, to establish a simple and reliable preservation method for the maintenance of stock Trichoderma harzianum strains isolated from soil cultivated with horticultural crop. Ten fungal isolates of T. harzianumwere selected for estimating the effect of different preservation methods on viability over time. The following preservation methods were compared: (1) distilled water stasis, (2) mineral oil, (3) silica gel, and (4) cellulose filter paper. Five replicates of the same T. harzianum strain were used for each storage method. For the distilled water stasis method, sterile screw-cap glass tubes measuring 1.5 15mm containing 4ml of sterile distilled water were used. The sporulating cultures on potato dextrose agar (PDA) were cut into small blocks (approximately 5mm) and placed into the tubes that were then kept at 4 1C. Cultures were recovered from storage by aseptically transferring a block of the inoculum from the water onto a fresh plate of 9ml of PDA. For storage in mineral oil, heavy mineral oil (Sigma Chemical Co., St. Louis, MO) double sterilized by autoclaving at 24h intervals was used. Slants (100 10mm) containing PDA and sporulating culture of fungi were covered with 5ml of mineral oil. Cultures were recovered from mineral oil storage by removing small blocks of the stored culture and transferring the blocks to fresh PDA. For storage with silica gel, uncolored, 200 mesh size, grade 40 silica gel (Fisher Scientific, Fair Lawn, NJ) was used after sterilization in an oven at 160 1C for 6h. Glass tubes (100 10mm) and caps were sterilized. Fungal spores were harvested by scraping the surface of a culture maintained in PDA with a sterile loop, and spore suspensions were made with sterilized distilled water (1 10 spores/ml). One hundred microliters of each suspension was placed into each tube containing 5g of silica gel that had been kept at 4 1C. The silica gel was mixed with the suspension by rotating the tubes several times until the crystals were easily separated. Then the tubes were maintained at 4 1C. Cultures were recovered by aseptically placing a few crystals onto fresh PDA. In the cellulose filter paper method, this substrate was used for fungal colonization. Ten sterilized cellulose filter paper strips measuring 20 10mm were put into each culture of T. harzianum. After the filter papers were well-colonized by the fungus, they were dried at room temperature for 5–7 days and stored in sterilized envelopes at 20 1C. The viability of all isolates was evaluated when the storage began as well as after 3, 6, 12, 18, and 24 months of storage. The fungal subcultures were evaluated on the basis of whether or not they grew after the different storage regimes at each time interval, and if they kept their original morphologic characteristics. All strains tested survived up to 24 months of storage with the methods 1, 2, and 4, but with method 3, 6 strains survived only to 12 months and 3 only up to 18 months. All strains recovered on PDA agar after 24 months storage in each of the methods used were evaluated for preservation of their original macroscopic and microscopic features. Strains recovered from materials stored in distilled water, mineral oil, and cellulose filter paper kept the original macroscopic characteristics. Assessment of the microscopic structures observed in microcultures indicated no significant changes between isolates retained by the different methods tested. This is the first report of the use of cellulose filter paper as a method of preservation of T. harzianum strains. We recommend the use of this method for Trichoderma strains preservation because it is simple, inexpensive, the least labor intensive, and requires very little refrigeration for the storage of the isolates.