Term paper of microbial physiology and metabolism Trichoderma harzianum

Submitted by:-SHASHI SHARMA

Roll no.-15Section –p8003

Reg. no.-11006142Msc. microbiology

<=Contents=>

1) Introduction of the Trichoderma harzianum

1.1) Taxonomy and genetics

1. 2) Mycoparasitism

2) Useful activity of Trichoderma harzianum

2.1) biocontrol activity.

3) Experiment showing the effects of Trichoderma harzianum

4) Role of T. harzianum in plants

4.1) in plant growth regulator4.2) secretion of endo-ch

5) Trichoderma harzianum SK-55 fungus, fungicide containing it, and method of manufacture of the same and its use.

6) Role in reduction of plant diseases

6.1) biological control of Rhizoctonia solania in tomatoes

7) Green house assays.

8) Field assays

9) Biological and integrated controls of Botrytis cinerea on apple with Trichoderma harzianum:-

10) Health consideration.

11) Environment consideration

12) Value consideration

13) References

Trichoderma harzianum:-

Trichoderma harzianum is a fungus that is also used as a fungicide. It is used for foliar application, seed treatment

and soil treatment for suppression of various disease causing fungal pathogens. Commercial biotechnological

products such as 3Tac have been useful for treatment of Botrytis, Fusarium and Penicillium   sp.

Taxonomy and genetics:-

Most Trichoderma strains have no sexual stage but instead produce only asexual spores. However, for a few strains

the sexual stage is known, but not among strains that have usually been considered for biocontrol purposes. The

sexual stage, when found, is within the Ascomycetes in the genus Hypocrea. Traditional taxonomy was based upon

differences in morphology, primarily of the asexual sporulation apparatus, but more molecular approaches are now

being used. Consequently, the taxa recently have gone from nine to at least thirty-three species.

Most strains are highly adapted to an asexual life cycle. In the absence of meiosis, chromosome plasticity is the

norm, and different strains have different numbers and sizes of chromosomes. Most cells have numerous nuclei, with

some vegetative cells possessing more than 100. Various asexual genetic factors, such as parasexual

recombination, mutation and other processes contribute to variation between nuclei in a single organism (thallus).

Thus, the fungi are highly adaptable and evolve rapidly. There is great diversity in the genotype and phenotype of

wild strains.

While wild strains are highly adaptable and may be heterokaryotic strains used for biocontrol in commercial

agriculture are, or should be, homokaryotic. This, coupled with tight control of variation through genetic drift, allows

these commercial strains to be genetically distinct and nonvariable. This is an extremely important quality control item

for any company wishing to commercialize these organisms.

Habitat of Trichoderma haziarnum:-

Trichoderma spp. is fungi that are present in nearly all soils. In soil, they frequently are the most prevalent culturable

fungi. They also exist in many other diverse habitats.

Trichoderma readily colonizes plant roots and some strains are rhizosphere competent i.e. able to grow on roots as

they develop. Trichoderma spp. also attacks, parasitize and otherwise gain nutrition from other fungi. They have

evolved numerous mechanisms for both attack of other fungi and for enhancing plant and root growth. Different

strains of Trichoderma control almost every pathogenic fungus for which control has been sought. However, most

Trichoderma strains are more efficient for control of some pathogens than others, and may be largely ineffective

against some fungi.

Trichoderma spp. continues to be a major source of contamination and crop loss for mushroom farmers.

Useful activity of trichoderma harzianum:-

In vitro biocontrol activity of Trichoderma harzianum on Alternaria alternata in the presence of growth regulators

The in vitro biocontrol ability of Trichoderma harzianum on the phytopathogen Alternaria alternata improved in the presence of the growth regulators gibberellic acid (GA3), or indolacetic acid (IAA) or benzylaminopurine (BAP) or foliar nutrient at concentrations similar or higher than those used at the field level. These plant hormones decreased the secretion of endopolygalacturonase (endo-PG) of A. alternata by approximately 20%, did not modify endochitinase (endo-CH) secretion of T. harzianum and did not alter germination of conidia or mycelia growth of any of these fungi. The presence of T. harzianumdecreased endo-PGase secretion of A. alternata by about 50%. This inhibitory effect was independent of the presence of growth regulators. The level of secreted endo-PG of T. harzianum was not modified by the presence of A. alternata, but the presence of this phytopathogen in cultures of T. harzianum, increased both the growth of the biocontroller and its secretion of endo-CH.

Trichoderma harzianum as a biocontrol agent:-

Trichoderma harzianum is an efficient biocontrol agent that is commercially produced to prevent development of several soil pathogenic fungi. Different mechanisms have been suggested as being responsible for their biocontrol activity, which include competition for space and nutrients, secretion of chitinolytic enzymes, mycoparasitism and production of inhibitory compounds of T. harzianum could be affected by environmental cues that include among others, the presence of plant nutrients at the field level, which contain growth regulators as auxins in their formulations. The presence of growth regulators

in the soil could come from applications done to the foliar portion of plants or to fruits, where part of applications fall either directly or from plants into the soil. They could also come from treatments of tubers with auxins, which are used to stimulate budding after seeding; or from soil microorganisms. In fact, it has been described that some fungal pathogens are able not only to induce increased levels of IAA in their respective hosts, but are themselves capable of producing IAA which is directly released into the soil. Agro bacterium tumefaciens, which causes crown gall on more than one hundred plant species, produces and contains genes coding for IAA and cytokinin production. Other bacteria, such as Pseudomonas savastanoi, code for IAA synthesis not only in genes contained in its plasmid but also in its chromosome. In addition, the direct use of cytokinins has been also described to reduce virus multiplication and local lesions in several plant species. Therefore, it seemed important to analyze if commonly used plant growth regulators such as auxins, cytokinins and gibberelic acid, that are applied to different plant species or that are produced by soil microorganisms, could affect the biocontrol activity of T. harzianum.

Alternaria diseases are among the most common diseases of many plants throughout the world. They affect primarily the leaves, stems, flowers and fruits of annual plants, especially vegetables and ornamentals. Alternaria alternata is a pathogenic fungus that can secrete endo-polygalacturonase (endo-PG) and pectate lyase (PL) activities. These enzymes are responsible for the hydrolysis of pectic components of the plant cell wall. Depending on the type of interaction developed between the plant species and the microorganism . These pectinases could represent one of the fungal infection mechanisms, or could be considered within the enzyme systems that trigger an hypersensitive response through the release of oligosaccharides acting as elicitors of the plant response. The activity of endo-PG and of PL is known to be affected by fungicides, temperature, metal ions or foliar nutrients. These latter products may contain growth regulators, or be applied together with them, which could also affect the activity of these enzymes. A. alternatawe decided to use this fungus as the target microorganism to test the biocontrol activity of T. harzianumin the presence or absence of phytohormones. A deepest knowledge of the factors that could affect biocontrol activity of Trichoderma would allow us to improve biocontrol conditions for trials at the field level, and to provide information of how hormones could benefit or decrease the biocontrol effect of T. harzianum in its specific interaction with A. alternata

Experiment showing the effect of theT.harzianum in biocontrolling agent:-.

Materials and methods :-

Chemicals. All chemicals like commercial plant growth regulators and plant nutrients distributed

Fungal isolates. A. alternata and T. harzianum were isolated from sooty molds infecting Citrus species in Chile and were maintained on potato dextrose agar.

Evaluation of mycelium growth and of biocontrol activity. Disks (0.5 cm diameter) from pure cultures of A. alternata or T. harzianum were seeded in a Petri dish containing either PDA or Mandels pectin agar MPA,, with or without the addition of 15, 30 or 40 ppm of gibberellic acid (GA3) or indolacetic acid (IAA) or benzyl amino purine (BAP), or of 15, 30 or 40 ppm of the commercial products mentioned in chemicals. Fungi were grown up to one week at 28°C. Colony diameter was recorded every two days. When biocontrol activity was tested, both A. alternata and T. harzianum were seeded in the same dish at opposite sides (dual cultures), and their growth was evaluated as above. Controls were performed seeding each fungus against itself. Results correspond to the mean of six different experiments run in duplicates.

Evaluation of conidia germination. Conidia from A. alternata or T. harzianum were obtained as recommended by AOAC. One hundred conidia from each fungus were grown on 2% (w/v) water agar (WA) for 18 hours in the absence or presence of 15, 30 or 40 ppm of GA3 or IAA or BAP or of the commercial products mentioned in chemicals. Germination was evaluated by light microcopy. Results correspond to the mean of five different experiments run in triplicates.

Production of endo-polygalacturonase (endo-PG) and of endo-chitinase (endo-CH) in submerged cultures. The secretion of the enzymes was evaluated in submerged cultures using the Mandels mineral salt medium (Mandels et al. 1974) with the addition of 4 g/l of Citrus pectin or glycol chitin for production of endo-PG or endo-CH, respectively. Flasks, containing 200 ml of any of the above media were inoculated with 1 x 106 conidia of either A. alternata or T. harzianum in the absence or presence of 15, 30 or 40 ppm of GA3 or IAA or BAP or the commercial formulations mentioned in chemicals.

To test the effect of T. harzianum on the ability of A. alternata to secrete endo-PG, 0.5 x 106 or 1 x 106conidia of each fungus were inoculated in the pectin-containing medium (200 mL), to give a final concentration of 1 x 106 or 2 x 106 conidia per flask.

To test the effect of A. alternata on the ability of T. harzianum to secrete endo-CH, 0.5 x 106 or 1 x 106conidia of each fungus were inoculated in the glycol chitin-containing medium (200 mL), to give a final concentration of 1 x 106 or 2 x 106 conidia per flask. Flasks were incubated at 28°C up to fourteen days with shaking at 150 rpm. The whole medium was then centrifuged at 9,000 x g for 10 minutes to remove mycelia, and the supernatant was used to test endo-PG or endo-CH activity. Results correspond to the mean of three independent experiments run in triplicates. Controls were performed with heat inactivated conidia. Data was analyzed through the test of Student at p<0.05.

Endo-polygalacturonase (endo-PG) activity. It was tested by a modified Nelson-Somogyi assay. The reaction mixture contained 0.5% (w/v) polygalacturonic acid in 100-mM sodium acetate pH 5.2. One unit was defined as the amount of enzyme that released 1 mM of reducing sugars per minute. The direct effect of GA3 or IAA or BAP on endo-PG activity was tested adding to the assay media of submerged cultures without additions, 15, 30 or 40 ppm of the corresponding growth regulator or of the commercial products mentioned in chemicals. Controls were performed with boiled enzyme. Results correspond to the mean of three different experiments run in triplicates.

Endo-chitinase (endo-CH) activity. Endochitinase activity was tested by the method of Pan et al. (1991) modified as follows: multi-well plates (20 mm diameter per well) were filled with 2% agarose containing 1% glycol chitin with or without the addition of 40 ppm of GA3 or IAA or BAP or of the commercial products mentioned in chemicals and were seeded with A. alternata, with T. harzianum or with the two fungi. Plates were incubated at 28°C for 72 hours, and total endo-CH activity was developed as described. Hydrolysis diameters were measured with a millimeter ruler. Results correspond to the mean of three experiments run in triplicates.

Result and discuss:-

Effect of plant growth regulators on the germination of conidia, on the growth of A. alternate and of T. harzianum, and on the biocontrol of A. alternata by T. harzianum

GA3 or IAA or BAP or commercial formulations of hormones, at concentrations of 15 or 40 or 80 ppm did not affect germination of conidia or fungal growth. In fact, 10 + 1% of conidia from A. alternata and 90 +10% of conidia from T. harzianum were germinated after 18 hours at 25°C in the presence or absence of the mentioned growth regulators. The presence of the foliar nutrient Bayfolan did not affect T. harzianumgermination but slightly increased that of A. alternata probably due to the presence of macro and micro elements in the formulation. On the other hand, the 2.4 + 0.2 cm growth of A. alternata and 8.8 + 0.2 cm growth of T. harzianum on PDA or MPA, after five days at 28°C, were not altered by the presence of any of the growth regulators or the foliar nutrient at the concentrations tested . These results

suggest that these plant growth regulators, either alone or included in the foliar nutrient formulation, may not affect germination and/or growth of the phytopathogen A. alternata or of the biocontrol agent T. harzianum at the field level, as a consequence of their use for the improvement of crop growth and productivity.

The in vitro biocontrol activity of T. harzianum was not affected by the presence of the growth regulators or the foliar nutrient. In fact, a 20% decrease in A. alternata development was observed in dual cultures both in the absence and in the presence of the hormones, suggesting that the inhibition of growth of A. alternata was due to the presence of T. harzianum. Also, an increase in the growth of T. harzianum was observed in these dual cultures, probably induced by the presence of A. alternata because none of the growth regulators or the foliar nutrient at the concentration used, altered this growth. Therefore, it could be expected that the use of plant growth regulators or formulations that contain any of these hormones at the field level would not affect the ability of T. harzianum to antagonize this fungal pathogen. Also, it appears that the presence of components added to commercial formulations of growth regulators or foliar nutrient do not alter the behavior of these fungi or of the phytohormones.

Role of T. harzianum in plants:-

Effect of plant growth regulators and of T. harzianum on the secretion of endo-PG from A. alternata

The maximal secretion of endo-PG from A. alternata into submerged cultures was reduced in 20 - 25% when this phytopathogen was grown in the presence of 40 ppm of GA3, IAA, or BAP. These results suggest that the hormones may interfere the secretion process of the enzyme or its levels, because none of the growth regulators tested decreased endo-PG activity of control. A stronger inhibitory effect was observed in the presence of active T. harzianum, which may be explained as a consequence of the growth inhibitory effect of the biocontrol agent on A. alternata. Due to the fact that T. harzianum also secretes endo-PG, although the enzymatic levels are much smaller than those secreted by A. alternata, it was necessary to determine if endo-PG secretion by T. harzianum was altered by the presence of the phytopathogen. No changes were observed in enzyme activity at day of maximal secretion of endo-PG by T. harzianum as a consequence of the presence of A. alternata. On the other hand, and opposite to the effect on this phytopathogen, the sole presence of growth regulators in culture media resulted in an increase of endo-PG secretion by T. harzianum. Endo-PG activity was not modified by the addition of the growth regulators at the assay medium of control, confirming the effect of these hormones on endo-PG secretion. It has been demonstrated that endo-PG from the biocontrol agent T. harzianum are participating in the release of oligogalacturonide elicitors from Citrus Limon cell walls during the development of the hypersensitive response. These may be concentrated into elicitor-active molecules through the formation of complexes between endo-PG and PGIPs.Therefore, the presence of growth regulators would stimulate one of the mechanisms for plant defense along with the ability to control fungal pathogens. Then, it may be suggested that during antagonism T. harzianumaffects cellular mechanisms in A. alternata that result in a slow down in its development and in a decrease of its infectious ability based on the levels of secreted endo-PG.

Effect of plant growth regulators and of A. alternata on the secretion of endo-CH from T. harzianum

The presence of 40 ppm of GA3, IAA or BAP did not affect the ability of T. harzianum to secrete endo-CH. A. alternata slightly increased the secretion of total endo-CH activity of the biocontrol agent, suggesting that the presence of the pathogen could serve as an additional inducer of this fungal cell wall degrading enzymes. Then, the presence of this pathogen would stimulate one of the mechanisms T. harzianum uses for its biocontroller activity. The differential expression of isoenzymes of endo-CH by Trichoderma has been described during the mycoparasitism of this fungus on pathogens, thus accounting for its antagonism against several fungal pathogens.

Trichoderma harzianum SK-55 fungus, fungicide containing it, and method of manufacture of the same and its use

The fungus Trichoderma harzianum SK-55 provides broad antagonistic interaction against plant pathogenic diseases, and may be used to control fungal diseases in plants. A fungicidal composition contains Trichoderma harzianum SK-55 isolated from the soil.

A method of manufacturing a fungicidal composition comprises introducing a large quantity of Trichoderma harzianum SK-55 into a culture medium, incubating it on the culture medium at a specific temperature range and at a specific humidity for a specific period of time, drying it at a specific low temperature range, and, if necessary, milling it into specific grain sizes. The fungicidal composition containing the incubated fungus may be distributed at the rate of 0.5 g to 5 g/m2.

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in

order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol

agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as

the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with

diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence

in Trichoderma-treated plots did not differ significantly from the control. A mixture of T.

harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this

case control was always significant (P=0·05) but improvement of control compared with each

treatment alone was not significant (P=0·05). The alternation of sprays with the biocontrol

preparation and with a dicarboximide fungicide was tested in three out of the five experiments

and was found to be effective, thus enabling a reduction in the use of chemical sprays.

Role in reducing plant diseases:-

Biological control of Rhizoctonia solani in tomatoes with Trichoderma harzianum mutants

Biocontrol of Rhizoctonia solani in tomatoes cultivated under greenhouse and field conditions was analyzed using the Trichoderma harzianum mutants Th650-NG7, Th11A80.1, Th12A40.1, Th12C40.1 and Th12A10.1 and ThF2-1, respectively. Their innocuousness on tomato cultivars 92.95 and Gondola (greenhouse assays), and on cultivar Fortaleza (field assays) was established. Alginate pellets (1.7 g pellets/L soil) containing c.a1 x 105 colony forming units (cfu)/g pellet were applied to a soil previously inoculated with R. solani at transplant (greenhouse) or to a naturally infected soil (field). Controls considered parental wild strains, a chemical fungicide and no additions. Mortality reduction was reflected in canker level lessening and in plant parameters increases. A different degree of susceptibility of tomato plants was observed, being Gondola cv. more resistant than 92.95 cv. to infection in a soil previously

inoculated with R. solani. Tomato plants of cv. Fortaleza did not show mortality in naturally infected soils (field assays), where the mutant ThF2-1 reduced significantly the canker level caused by R. solani.

R. solani is one of the phytopathogens that attack tomatoes cultivated under greenhouse conditions, causing root and crown rot. R. solani is controlled using methyl bromide, a fumigant known for its high toxicity and its degradative effect on the ozone layer.

Fungi of the genusTrichoderma are important biocontrol agents (BCAs) of several soil borne phytopathogens .Trichoderma use different mechanisms for the control of phytopathogens which include mycoparasitism, competition for space and nutrients, secretion of antibiotics and fungal cell wall degrading enzymes. In addition, Trichoderma could have a stimulatory effect on plant growth as a result of modification of soil conditions.

The biocontrol by fungal species of the Trichoderma genre, of root and crown rot caused by R. solani, are being used as an alternative to chemical fungicides. Nevertheless, the need to improve the positive effect of wild BCAs has prompted the development of different strategies, including those related to increasing the expression and secretion of enzymes such as chitinases and endoglucanases involved in the degradation of the phytopathogen’s cell walls. This strategy has resulted in mutant strains with enzyme activities higher than those of their wild-type and where T. harzianum mutant strains have proved to be effective in vivo biocontrol agents of R. solani that causes crown and root rot in tomatoes. However, their innocuousness on tomato plants and their effectiveness as biocontrol agents on R. solani has not yet been tested.

Greenhouse assays: effectiveness of native and mutant Trichoderma strains in the control of R. solani in greenhouse tomatoes

Sodium alginate pellets of the wild or mutant strains of T. harzianum innocuous for these tomato cvs. at the concentrations indicated in Table 1, were prepared) and 4 g/pot of each strain were applied at transplant to the soil . They were mixed with the soil close to where the root pan and crown of the plant were going to be planted. Plants were grown under greenhouse conditions, where soil temperature ranged from 20.9ºC to 28.6ºC and watered without fertilizers. Each experimental unit corresponded to one plant of each cultivar per each of the nine treatments, considering five replications. Pots were arranged in a complete random design. Once plants reached fruit set between the fourth and the fifth bunch, they were removed, the potting soil was rinsed from the roots and plants were assessed as follows:

a) Crown canker (area size showing lesions in crown related to stem perimeter), was assessed using the following scale, to establish the disease’s degree: 0: 0% area affected, healthy plant; 1: < 1% area affected, slight disease; 2: 5% - 30% affected area, moderate disease; 3: 30% - 60% area affected, important disease; 4: 60% - 90% area affected, severe disease; and 5: > 90% area affected, dead plant.

b) Root development was evaluated using the following scale: 0: no development, dead plant; 1: poor development, weak plant; 2: moderate development, stable plant; 3: good development, healthy plant; and 4: very good development, healthy plant.

c) Plant mortality was estimated using crown canker and root development results.

d) Aerial fresh and dry weight, and root fresh weight were assessed by weight.

Measured parameters in weight units were analyzed by ANDEVA, and when significant differences were detected, Tukey’s test was used at 5% significance. Crown canker and root development results were analyzed using the non-parametric Kruskal-Wallis test, and when significant differences were obtained, the Mann-Whitney pair’s comparison was used.

Field assays: effectiveness of wild and mutant Trichoderma strains in the control of R. solani under field conditions:-

Field assays were run under a commercial producer cold greenhouse, located in Olmue (V Region, Chile), using tomato plants cv. Fortaleza, in the season winter - spring. A high incidence of R. solani in previous seasons in this field was considered for its selection to run the assay.

Treatments considered the use of two mutant strains: Th12A10.1 and ThF2-1 , previously selected as good R. solani controllers in vitro . These strains were formulated as alginate pellets and their effect was compared with the commercial fungicide Trichonativa (Trichoderma harzianum strain Queule, Trichoderma virens strain Sherwood and Trichoderma parceanamosum strain Trailes, and with the fungicide methyl bromide. An additional control was run with sodium alginate pellets that did not contain any biocontrol agent. The natural Trichoderma spp. population was established before the assay.

a) To the planting hole in the soil pre-transplanting of tomato plants in one inoculation assays.

b) To the planting hole in the soil pre-transplanting of tomato plants, around the crown 15 days after transplanting and one week before sprout in three inoculation assays.

c) In the watering following the recommendations of the manufacturer (5 cc/L) in the planting whole pre-transplanting and one L/Ha in the following inoculations of the commercial product (Bioinsumos Nativa, S. A.).

Effectiveness of trichoderma in tomato plant improvement:-

All the native and mutant Trichoderma strains tested were innocuous to tomato seedlings and plants of cvs. 92.95 and Gondola confirming the innocuousness of several of these Trichoderma strains already reported, for experiments run with tomato plants in different conditions. Therefore, they were used in greenhouse and field assays to test their biocontrol effect on R. solani.

The general effectiveness of wild and mutant strains of T. harzianum on the biocontrol of R. solani disease on tomato plants cv. 92.95 can be visualized in Figure 1. Control plants (T0), and plants with Th12 and Th650 treatments showed death and scarce root development, respectively; while the others appear healthy showing different degrees of development, depending on the treatment shown in Table 1. Analysis of plant mortality. 100% mortality caused by R. solani was reduced to zero by the mutant Trichoderma strains Th650-NG7, Th12A10.1 and Th11A.80.1, while in the presence of Th11C40.1 and Th11 the biocontrol effect was identical to the treatment with the chemical fungicide. The increase in survival caused by the parental strains Th650 and Th12 was lower than the one caused by the corresponding derived mutant. The same mutants showed the lowest canker level with no significant differences between them, although Th650-NG7 and Th12A.10 were not different from Th11 and with the treatment with pencycuron (T1). In general, the canker level observed in the different treatments, correlate well with results in mortality and those of root development, where treatments with Th650-NG7, Th12A10.1 or Th11A.80.1 resulted in the highest root development. These results suggest that mutants obtained either by chemical or UV treatments, with the exception of Th11C40.1, have improved their biocontrol activity in relation to their parental strains. The obtainment of Trichoderma mutants through different procedures has allowed the obtainment of hyper hydrolytic strains with improved biocontrol activity on R. solani , of protease overproducing strains with improved biocontrol activity against different pathogens, of salt tolerant strains for the control of F. oxysporum and of pesticide-polyresistant strains, thus supporting the improved biocontrol effect of some of the mutants tested in this work.

The decrease in % mortality and in canker level and the increase in root development of tomato plants cv. 92.95 caused by Th650-NG7, Th12A10.1 and Th11A.80.1 was also observed in plant fresh weight and root dry weight, where treatments with these mutants resulted in the highest values, although Th12A10.1 was the only mutant that caused significant differences in all parameters evaluated. The statistical analysis of results shows that the aerial fresh weight caused by the presence of Th650-NG7 doubles that of Th650, although the latter showed no significant differences with T0. These results do not agree with those previously reported for tomato plants cv. Cal Ace, where assays were run using soil fumigated with methyl bromide rather than sterile soil. The best results on tomato plants cv. Cal Ace were obtained with the wild strain Th650, using an inoculum three times lower than the one used in this assay. The lower inoculum used with tomato cv. Cal Ace could explain in part the differences observed among assays, but it cannot be discarded genotypic differences among cultivars in their response to the presence of the same fungal strain or a deleterious effect of the Th650 dose used in this assay (Table 3).

Fresh weight and fruit yield of tomato plants cv. Fortaleza: effect of mutants under field conditions

Fresh plant weight did not differ between treatments which agrees with results obtained in greenhouse assays for cv. Gondola inoculated with R. solani , and also with the moderate degree of the disease caused by the phytopathogen in the field experiment.

On the other hand, the use of the two mutant Trichoderma strains did not result in an improved weight of tomato plant roots, which neither showed higher weights in the presence of the commercial product. These results are different from those of who obtained higher dry weight of tomato plants treated with the BCAs than controls, in assays of tomato plants grown in soil infected by Pyrenochaeta lycopersici where the growth promoting effect was attributed to the presence of the BCAs, as already reported.

Fruit yield, related to second and third quality fruits did not show differences between treatments, and first quality fruits showed significant differences between treatment with methyl bromide and all the other treatments. As methyl bromide destroys all microorganisms present in soil, independently of their beneficial or damaging activity; it is not unexpected that yield of first quality fruits was lower in this treatment because of the lack of beneficial organisms in the soil that contributes to plant nutrition. On the other hand, treatments with Trichoderma did not cause significant differences in yield of first quality fruits, suggesting that the presence of BCAs had no effect on this parameter, which in turn was similar to controls where soil contained only the natural micro flora.

Damage caused by R. solani in tomato plants cv. Fortaleza under field conditions and different treatments (Figure 5) shows that the mutant ThF2-1 strain showed the lowest level of damage as opposed to controls. The damage level caused by R. solani on the control tomato plant cv. Fortaleza of 1.58, lower than those obtained for cv. 92.95 of 4.8, and for cv. Gondola of 2.8, suggests that the level of resistance could rely on the genotypic characteristics of each cultivar. The differences in resistance in terms of damage level caused by R. solani 618, observed between the tomato cv. tested are also reflected in the differences in plant mortality.

Total tomato fruit yield in plants treated with the mutant ThF2-1 showed no significant differences with other treatments, suggesting that damage level caused by R. solani is not related to this parameter, at least in this tomato cv. On the other hand, the three applications of ThF2-1 improve total tomato fruit yield when compared to the single pre-transplanting application. Results obtained with ThF2-1, a strain obtained through protoplast fusion agree with the fact that biocontrol strains obtained by these means would contain more and/or more effective biocontrol mechanisms as result of hybridizations between different strains.

The lowest fruit yield was obtained with methyl bromide treated plants, most probably due to a phytotoxic effect and/or to an inadequate application of the chemical fungicide.

Biological and integrated controls of Botrytis cinerea on apple with Trichoderma harzianum:-

Biological and integrated controls of dry eye rot on apple caused by Botrytis cinerea were possible under natural field conditions by the use of the antagonistic fungus Trichoderma harzianum. A fungicide-resistant isolate of T. harzianum, P1, controlled dry eye rot better than the fungicide-sensitive parent strain both alone and together with reduced dosages of the fungicide vinclozolin. Although integrated control was not significantly more effective than biological control or chemical control by itself, the combination of two different control methods may provide a more consistent control and reduce the risk of fungicide-resistance developing in the pathogen. Use of fungicide-resistant strains of the biocontrol agent is also necessary as long as other diseases on the crop are controlled by fungicides.

Disease Control and Pest Management

Prolonged exposure of mycelia and conidia of Trichoderma harzianum to the fungicide benomyl did not produce isolates tolerant to the fungicide. Exposure of four wild strains of T. harzianum to the fungicides chlorothalonil, procymidone, iprodione, and vinclozolin resulted in selection of several isolates tolerant to these fungicides. Some of the fungicide-tolerant isolates grew better radially on media containing the fungicides than their respective wild strains did. Other isolates lost their tolerance after being cultured on fungicide-free media. Conidia of certain isolates of the wild strains WT-6 and T, tolerant to chlorothalonil and iprodione, respectively, germinated better on media containing high concentrations of the fungicides than did conidia of their respective wild strains. Exposure of conidia of the wild strain T-14 to 0.1% (active ingredient) chlorothalonil for 4 wk reduced germination by 80% when the conidia were placed on a fungicide-free medium. Similar exposure of conidia of T-14(3M), a chlorothalonil-tolerant isolate, reduced germination by only 20%. An iprodione-tolerant isolate derived from the Egyptian strain T produced more toxin, as measured by inhibition of mycelial growth of Sclerotium cepivorum, than did the wild strain. One fungicide-tolerant isolate of strain Th-1 (Th-1 [procy-2M]) reduced white rot of onion caused by S. cepivorum more effectively than did Th-1 or other fungicide-tolerant isolates. The iprodione-tolerant isolate T (ipro-25M) and iprodione combined with T(ipro-25M) gave the best control of white rot of onion in the field in Egypt.

Health consideration:-

Trichoderma harzianum Rifai strain KRL-AG2 is unlikely to affect your health when used according to the label directions.

Exposure to T. harzianum Rifai strain KRL-AG2 may occur when handling of Root Shield Drench Biological Fungicide Wettable Powder or Root Shield Granules Biological Fungicide. When assessing health risks, several key factors are considered: the microorganism's biological properties (e.g. production of toxic by-products), reports of any adverse incidents, its potential to cause disease or toxicity as determined in toxicological studies and the likely levels to which people may be exposed relative to exposures already encountered in nature to other strains of the microorganism.

Toxicology studies in laboratory animals describe potential health effects from large doses in the hope of identifying any potential to cause disease or toxicity. No significant toxicity and no signs of causing diseases were observed when T. harzianum Rifai strain KRL-AG2 was tested on laboratory animals.

Residues in Water and Food

Dietary risks from food and water are not of concern.

Pesticide maximum residue limits (MRLs) are established for Food and Drugs Act purposes through the evaluation of scientific data under the Pest Control Products Act. Each MRL value defines the maximum concentration in parts per million (ppm) of a pesticide allowed in or on certain foods. Food containing a pesticide residue that does not exceed the established MRL does not pose an unacceptable health risk. The Food and Drugs Actprohibits the sale of adulterated food, that is, food containing a pesticide residue that exceeds the established MRL.

Trichoderma harzianum is common in most terrestrial environments, and the use of Root Shield Drench Biological Fungicide Wettable Powder and Root Shield Granules Biological Fungicide is not expected to significantly increase the natural environmental background levels of this microorganism. Furthermore, Trichoderma species are rarely reported to occur on living plants. Based on the use pattern for Root Shield Drench Biological Fungicide and Root Shield Granules Biological Fungicide, minimal dietary exposure to residues of T. harzianum Rifai strain KRL-AG2 and its secondary metabolites are expected. No adverse effects have been reported for this microbial pest control agent in the United States where it has been registered for use since 1990. In addition, no significant toxicity or signs of causing disease were observed when T. harzianum Rifai strain KRL-AG2 was administered orally to rats. Therefore, establishment of an MRL is not required for T. harzianum Rifai strain KRL-AG2. The likelihood of residues of T. harzianum Rifai strain KRL-AG2 contaminating drinking water supplies is negligible to non-existent. Consequently, dietary exposure and risk are minimal to non-existent.

Environmental Considerations:-

Environmental risks are not of concern:-

Field studies suggest that T. harzianum Rifai strain KRL-AG2 will likely disperse and persist in the environment. Published reports indicate that there may be potential negative effects on non-target plants and beneficial soil microorganisms. However, application of Root Shield Drench Biological Fungicide Wettable Powder or Root Shield Granules Biological Fungicide is limited to specific food and non-food crops grown in greenhouses. Therefore, release to the environment and exposure and risk to non-target terrestrial and aquatic organisms is negligible.

A few adverse effect reports have identified T. harzianum as the causal agent of "green mould disease." Although the active ingredient's potential to cause this disease is unknown, a label statement preventing greenhouse operators from distributing treated plant matter to mushroom growers for use as substrate is required.

Value Considerations:-

What Is the Value of T. harzianum Rifai Strain KRL-AG2?

The end-use products Root Shield Drench Biological Fungicide Wettable Powder and Root Shield Granules Biological Fungicide are bio fungicides that contain the active ingredient T. harzianum Rifai strain KRL-AG2 for the suppression of fungal root diseases on tomato, cucumber and ornamental crops. The registration of these two products will provide the Canadian greenhouse industry with an additional non-chemical fungicide. The active ingredient T. harzianum Rifai strain KRL-AG2 employs multiple modes of action for the suppression of fungal pathogens and, therefore, can be used as a resistance-management tool.

Measures to Minimize Risk

Registered pesticide product labels include specific instructions for use. Directions include risk-reduction measures to protect human and environmental health. These directions must be followed by law.

The key risk-reduction measures on the label of Root Shield Drench Biological Fungicide Wettable Powder and Root Shield Granules Biological Fungicide to address the potential risks identified in this assessment are as follows.

Risk-Reduction Measure

Human Health

As a standard precaution anyone handling or applying Root Shield Drench Biological Fungicide Wettable Powder or Root Shield Granules Biological Fungicide must wear waterproof gloves, a long-sleeved shirt, long pants and shoes plus socks. In addition, a NIOSH approved respirator must be worn by applicators and early-entry workers. Early-entry workers will also be restricted from entering greenhouses for up to four hours following treatment of the crops with Root Shield Drench Biological Fungicide Wettable Powder or Root Shield Granules Biological Fungicide.

Environment

As a general precaution, handlers are advised not to contaminate irrigation or drinking water, or aquatic habitats while cleaning equipment or disposing of waste. In addition, the following label statement is required: "The treated plant material must not be used as substrate for mushroom farms."

Other Information

The relevant test data on which the decision is based (as referenced in this document) are available for public inspection, upon application, in the PMRA's Reading Room (located in Ottawa). For more information, please contact the PMRA's Pest Management Information Service by phone at 1-800-267-6315) or by e-mail at pmra_infoserv@hc-sc.gc.ca.

Any person may file a notice of objection5 regarding this registration decision within 60 days from the date of publication of this Registration Decision. For more information regarding the basis for objecting (which must be based on scientific grounds), please refer to the PMRA's website (Requesting a Reconsideration of Decision, http://www.pmra-arla.gc.ca/english/pubreg/reconsideration-e.html) or contact the PMRA's Pest Management Information Service by phone at 1-800-267-6315 or by e-mail at pmra_infoserv@hc-sc.gc.ca.

References:-

1) AL-KARAKI, Ghazi N. Growth, water use efficiency, and sodium and potassium acquisition by tomato cultivars grown under salt stress. Journal of Plant Nutrition, January 2000, vol. 23, no. 1, p. 1-8.

2) APABLAZA HIDALGO, Gastón. Patología de cultivos epidemiologic y control holístico. Santiago; Ediciones Universidad Católica de Chile. 2000. 347 p. ISBN 956-14-0561-X.

3)ARIAS, M.; HERRERA, R.; BESOAIN, X; PÉREZ, L.M. and MONTEALEGRE, J. Evaluations in vitro de mutantes de cepas de Trichoderma para el control de Rhizoctonia solani y Phytophthora nicotianae en tomato. Boletín Micológico, December 2006, vol. 21, no. 3.1, p. 71-75.

4)BENÍTEZ, Tahía; RINCÓN, Ana M.; LIMÓN, M. Carmen and CODON, Antonio. Bioncontrol mechanisms of Trichoderma strains. International Microbiology, December 2004, vol. 7, no. 4, p. 249-

260.

5)DUNIWAY, J.M. Status of chemical alternatives to methyl bromide for pre-plant fumigation of

soil.Phytopathology, December 2002, vol. 92, no. 12, p. 1337-1343.

6) GUTIÉRREZ, Bárbara; GONZÁLEZ, Maria S. and SALIH, Alberto. Caracterización de aislamientos deRhizoctonia solani (Kühn) que inducen pudriciones radicales en cultivares de caraota (Phaseolus vulgaris L.). Bioagro, 2006, vol. 18, no. 1, p. 63-72.

7) Asociación Nacional de Fabricantes e Importadores de Productos Fitosanitario Agrícolas (AFIPA).(1993-1994). Manual Fitosanitario. AFIPA, A.G. Gredos Ltda., Santiago, Chile. 623 p.

8) Association of Official Analytical Chemists (AOAC). (1980). Official methods of analysis of the Association of Official Analytical Chemists. Horwitz, H. ed. Arlington, Virginia, USA. 61p.

9)Aubá, M.; Chiong, M. and Pérez, L.M. (1993). Effect of foliar nutrients, fungicides, temperature and metal ions on pectate lyase and polygalacturonase from Alternaria alternata found in association with sooty molds on Citrus trees. Fitopatologia 28:38-44.