TRICHODERMA ATROVIRIDE
(COLD TOLERANT TRICHODERMA)

Dr. Jenifer Huang McBeath
Plant Pathology and Biotechnology Laboratory
University of Alaska Fairbanks
Fairbanks, AK 99775-7200
TEL: (907) 474-7431
FAX: (908) 474-7439
Email: ffjhm@uaf.edu

Background

       Trichoderma atroviride is a cold tolerant, versatile hyperparasite isolated from the sub-arctic region of Alaska. In Alaska, controlling diseases such as snow mold through the application of chemical fungicide is costly and often ineffective. Because of the fragility of northern environments, chemical use also elicits great concern. Activities of hyperparasites were noticed in the early 80's in studies of snow mold on winter wheat. It was found that the disease severity on wheat worsened when benomyl was used at half lethal dosage. The results indicated that additional intervening factors, mostly biological, were also present. T. atroviride CHS 861 was selected from more than 54 fungal and 400 bacterial hyperparasites isolates of soils collected from various plant communities in the state.

Characteristics of Trichoderma atroviride

       Trichoderma atroviride ,CHS 861 is a strong hyperparasite and can parazitize a wide range of pathogenic fungi. A temperature range of 4 C (and less) to 33 C makes it useful in controlling pathogens that can inflict damage on roots, stems and other plant tissues under cool temperatures when plant tissues are particularly vulnerable. T. atroviride can parasitize many economically important plant pathogens such as Armellaria mellea, Botrytis cinerea, Caprinus psychromobidus, Fusarium solani, Microdochum nivale, Myriosclerotinia borealis, Phytopaththora spp. (P. cactorum, P. capcisi, P. infestans, P. parasitica), Pythium spp. (P. aphanidermatum, P. dissotacum, P. ultimum), Rhizoctonia solani, Scleortinia sclerotiorum), Sclerotial Low Temperature Basidiomycetes, Typhula spp. (T. incarnata,,T. idahoensis, T. ishikariensis), and Verticillium dahliae.

       Trichoderma atroviride is fast growing and produces profuse spores. It is rhizosphere competent and possesses plant growth promotion abilities. T. atroviride CHS 861 is naturally resistant to metalyxyl (ridomil), captan and Pentachloronitrobenzene (PCNB, or terraclor), but is very sensitive to benomyl. To expend its usefulness, benomyl resistant mutants were also induced. T. atroviride biotypes (mutants) possess characteristics similar to the wild type, but is highly resistant to benomyl.

Potential Plant Pathogens and Diseases they caused affected by Trichoderma atroviride

Fungal Species                      Diseases Caused              Important Plant Hosts
Armillaria mellea                        root rot                         fruit trees, deciduous trees,
                                                                                           conifers

Botrytis cinerea                         grey mold                     cucumber, grapes,
                                                                                           strawberry, raspberry,
                                                                                           tomatoes, tree seedlings

Caprinus psychromobidus        snowmold                     rye, turf grass, wheat,
(=sclerotial Low Temperature
Basidiomycete)

Fusarium solani                          stem rot                        tomato, vegetable and
                                                                                            flowers

Microdochum nivale                   pink snow mold            rye, turf grass, wheat

Myriosclerotinia borealis           snow mold                    rye, turf grass, wheat,

Pythium spp.                               damping off                 ginseng, monocot and dicot
                                                                                            seedlings, leak, potatoes

Phytophthora infestans             late blight                     potato (as tuber and seed
                                                                                            pieces treatment)

Phytophthora capcisi                  blight, root rot            chili pepper, sweet pepper,                                                                                             eggplants

Phytophthora cactorum             stem rot                       ginseng, apple

Rhizoctonia solani                       black scurf                   potatoes
                                                      damping off                 corn, flowers, ginseng,                                                                                             vegetables
                                                      Sore shine                   cotton
                                                      Tip off                          ginseng, carrot

Sclerotinia sclerotiorum             stem rot                       rapeseed, flowers,
                                                                                            potatoes
                                                      white mold                   beans, carrots, crucifers

Sclerotium rolfsii                         southern blight            orchid, peanut, soybean,                                                                                             turf grass

Typhula idahoensis                     snow mold                   rye, turf grass, wheat

Typhula incarnata                       snow mold                  rye, turf grass, wheat

Typhula ishikariensis                   snow mold                  rye, turf grass, wheat

Verticillium dahliae                      wilt                               cotton, horse radish,
                                                                                            potato, fruit and
                                                                                            nut trees, strawberry

Effects of Trichoderma atroviride on Plants and Efficacy of Disease Control

      Trichoderma atrovirde show no phytotoxicity to plants. On the contrary, plants treated with T. atroviride demonstrated marked responses in growth-plants are bigger, healthier, and flower earlier. T. atroviride seems to forge a symbiotic relationship with plants. For instance, T. atroviride mycelia and chlamydospores and chlamydospore primordia were observed in the root cells of winter wheat with no adverse effects on the plants. T. atroviride-treated winter wheat plants had higher rates of survival against snow mold disease.

      For more than ten years, many field and greenhouse studies have been conducted testing the efficacy of T. atroviride in controlling economically important diseases caused by various plant pathogens.

a) Phytophthora blight of pepper and eggplants

      Phytophthora capsici can cause severe loss on eggplants growing under hothouse conditions. In the US, peppers, sweet and chili, are one of the most important vegetable crops affected by this pathogen. In the state of New Mexico, approximately 50,000 acres of chili peppers are cultivated annually. The most serious problem effecting pepper production is Phytophthora blight. This disease, caused by Phytophthora capcisi, reduces seed germination and causes damping off of seedlings, wilt of young plants and rot of fruit. Every year, approximately 40% of the crop is destroyed by this disease. Because there is no control for Phytophthora blight, pepper producers routinely plant 40% more acreage of peppers to compensate for the expected losses.

      In the efficacy studies conducted in our greenhouse, the application of T. atroviride to pepper seeds grown in P. capsici infested soils improved seed germination from 83% (blank control) to 100% (Trichoderma treatment at dosage 104 or 105 spores/ml). The survivability of pepper seedlings also increased from 50% (no Trichoderma treatment) to 100% (Trichoderma treatment at dosage 105 spore/ml). T. atroviride appeared to promote the growth and development of pepper seedlings. In the T. atroviride-treated plants, cotyledons were larger and the true leaves emerged earlier and expanded faster. These plant responses varied in direct correlation with the dosages. True leaves in the seedlings treated with 105 spores/ml emerged 14 days sooner than those of the blank control.

      Efficacy studies were conducted under field conditions in New Mexico.

b) Black scurf of potatoes

       Rhizoctonia solani can cause damping off on seedlings of flowers and vegetables, bottom rot of lettuce and other vegetables, root rot of corn, sore shin of cotton and bare patches of turfgrasses (a serious problem on golf courses). On potatoes, R. solani causes black scurf. This disease can cause yield reduction by producing fewer and/or malformed tubers. More importantly, black scurf on the surface of the potato tubers makes it unappealing to customers and reduces its value.

      Field trials to test the aptitude of T. atroviride in controlling R. solani on potatoes were conducted in Alaska (three years) and Montana (three years). The field trials were conducted on fields known to have a heavy infestation of R. solani. The experiments conducted in Alaska included wild types (CHS 861) and biotypes, a blank control and a PCNB fungicide control. Results of the field trials indicated that T. atroviride treatment improved the quality of potatoes considerably. A significantly larger amount of black scurf was found on potato tubers produced from the blank control (51%) than CHS 861 (29%), biotype 453 (7%), biotype 603 (28%) and PCNB (16%).

      In the field trials conducted in Montana, the treatment variables included wild types and biotypes, a carrier control, two blank controls, a chemical control (Tops MZ) and a bi-nucleate Rhizoctonia isolate. Significantly larger amount of black scurf was found on potato tubers produced from the blank control (73%) and carrier control (50%) treatments. Treatments of T. atroviride 861 (39%), 453 (39%), 603 (39%) and 901(27%) were comparable in their effectiveness in controlling black scurf to Tops MZ (35%) and bi-nucleate Rhizoctonia (34%).

c) Late blight of potatoes and tomatoes

       Late blight is the single most important disease of potatoes worldwide. It is destructive wherever potatoes are grown, except in dry areas. The Irish "potato famine" of the 1840s was caused by the A1 strain of Phytophthora infestans, causal agent of this disease. For over hundred years, this disease was control fairly well by the use of resistant varieties and by chemical fungicide applications. Ridomil, a systemic fungicide, was especially effective. In early 90s, an A2 strain of P. infestans was found in most of the potato growing areas in the world. Varieties that are resistant to A1 were found highly susceptible to the A2 strain. The A2 strain is highly resistant to Ridomil and many other chemical fungicides. Because it is sexually compatible with A1, hybridization between the two strains became possible. Currently, many new races that are highly resistant to chemical fungicides were found and make control of this disease increasingly difficult. Because of the lack of alternatives, an intensive fungicide application schedule is needed. In an extreme case of late blight disease control, thirteen (13) chemical applications were conducted in twelve (12) days.

       Furthermore, increasing evidence indicates that late blight can be transmitted through disease seed tubers and through seed pieces which become infested during the pre-cutting process. No effective seed pieces treatment is presently available.

       Trichoderma atroviride is an effective biological control agent of P. infestans. Similar to P. infestans, it also prefers cool, humid conditions for its growth. It parasitizes A1 and A2 strains equally well and is also resistant to Ridomil. T. atroviride is especially effective in controlling late blight on seed pieces. Efficacy studies indicated that Trichoderma atroviride treatment provided effective protection to potato seed pieces that is equal to or better than chemical fungicide control. The germination rate of potato seed pieces treated with T. atroviride was equal to those of blank control (100%) compared with un-treated, disease contaminated seed pieces (15%). The germination rate of seed pieces treated with chemical fungicides (fludioxinil +EBDC) treatment was 88%. Sprout initiation from seed pieces treated with T. atroviride seemed to be faster and produce a more even stand.

d) Damping off of seedlings

       Damping off disease can cause seeds to rot in the soil and seedlings to wilt and die. This disease affects numerous species of vegetables, flowers, melons, field crops and trees; among them, garden peas are the most susceptible. Many plant pathogens, including Phytophthora spp. and R. solani, can cause damping off disease, and Pythium spp. is by far the most prevalent.

       Trichoderma atroviride showed a significant ability to protect plants against damping off disease. In the efficacy studies conducted in the greenhouse, garden peas coated with T. atroviride spores were planted in Pythium spp. infested soils. A total of seven Pythium isolates, including P. aphanidermatum, P. dissotocum, P. violae, P. ultimum, were tested. Significant increases in the germination of pea seeds grown in Pythium-infested soils were observed in seeds coated with CHS 861 (66% germination) or biotype 603 (44% germination) when compared to the seeds without Trichoderma treatment (11% germination).

       Growth promotion characteristics have also been observed on T. atroviride-treated plants in the absence of detectable plant pathogens. Significant increases of weight, height as well as numbers of flowers and pots were found on T. atroviride-treated pea plants.

e) Sclerotinia stem rot of flowers

       Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is one of the most serious diseases on garden flowers in high latitude regions. The pathogen produces many large sclerotia on the surface and in the hollow centers of the stem. The sclerotia survive the harsh winter well; they can stay viable in the soil for many years. Each spring, these sclerotia serve as the primary inoculum of the disease. In many cases, this disease is so severe that it makes the cultivation of flowers, especially petunias, impossible. Control of this disease is extremely difficult. Systemic fungicide Topsin M showed some effects, but it is very costly. Furthermore, it does not have any effect on sclerotia.

       Results of laboratory tests indicated that T. atroviride is an effective hyperparasite of S. sclerotiorum. The hyphae of T. atroviride penetrates the hyphae of S. sclerotiorum and causes lysis of the pathogen. T. atroviride has been found to parasitize the sclerotia of S. sclerotiorum (as well as the sclerotia of other plant pathogens such as Botrytis cinerea, Verticillium dahliae) and use them as a food source.

       In the efficacy studies, treatment variables included T. atroviride in the potting mix, T. atroviride in potting mix and in field soils, Topsin M (applied as a drench) and a blank control. Petunia plants treated with Topsin M and T. atroviride stayed in bloom longer. These plants were also significantly healthier than the blank control, as measured by fresh weight and dry weight. Application of Topsin M and T. atroviride in potting mix as well as in field soils yielded significantly less sclerotia per plant.

f) Verticillium wilt of alfalfa, cotton, strawberry and fruit trees

       Verticillium wilt, caused by Verticillium dahliae, is a serious disease of alfalfa, cotton, potatoes, tomatoes, strawberries and fruit and nut trees. V. daliae is a slow-growing fungus but it is an extremely prolific producer of conidia and micriosclerotia which often cause blockage in the water conducting vessels (xylem) of plants and resulted in wilt and slow death of host plants. The most common means of control of this disease is to fumigate the soils with methyl bromide.

       Trichoderma atroviride has been found capable of utilizing V. dahliae as food sources. In the studies of hyperparasitism, clumps of T. atroviride conidia were dusted onto vigorously growing colonies of V. dahliae with numerous microsclerotia. No fungistatic effect to T. atorviride was ever observed from V. dahliae; T. atroviride conidia germinate readily on the surface of hypha of host fungi and their germ tubes penetrate into the hypha of V. dahlia. (Similar phenomena were found in other plant pathogens tested, including Botrytis cinerea, Rhizoctonia solani, C. psychromobidus.) Parasitism of T. atroviride resulted in the total arrest of the growth and development of V. dahliae-rapid loss of structural integrity of conidia, mycelia and microsclerotia of V. dahliae already formed and no formation of new microsclerotia. The ability of T. atroviride to cause deterioration of V. daliae conidia and microsclerotia could relieve the blockage of xylem and restore the vigor of plants.

g) Snow molds

       Snow molds are fungi which can attack plants under a cover of snow. They comprise over a dozen species, such as Caprinus pshchromobidus, Microdochium nivale, Myriosclerotinia borealis, Typhula incarnata, T. ishikariensis, T. idahoensis, occur commonly in high latitude regions. The pathogens infest host plants (winter wheat, winter rye, turfgrasses, etc.) in early winter when the soil is not yet frozen. During the long winters, under a thick snow layer, snow mold fungi proliferate and spread in host tissues under the dark, humid conditions. Death of the plant is due to maceration of plant tissues by enzymes of snow mold fungi and depletion of carbohydrate reserves. Snow molds are difficult to control. The application of fungicides is costly and often ineffective.

       Trichoderema atroviride has been found capable of utilizing Caprinus pshchromobidus, Microdochium nivale, Myriosclerotinia borealis, Typhula incarnata, T. ishikariensis and T. idahoensis as food source. Hyhae of T. atroviride pentrated freely through the cell walls and became interwined with the hyphae of the snow mold fungi. Cell deterioration and lysis occured rapidly. In M. boralis, the sclerotia not only provides a means of survival but also serves as its primary source of inoculum. T. atroviride has been found to be capable of parasitizing the sclerotia of M. borealis and using it as a food source.

       The efficacy studies of T. atroviride in controlling C. pshchromobidus were conducted under conditions of cold (about 4C), dark and 100% humidity for 8 weeks. Because of the extremely trying conditions, a certain degree of mortality was observed in all treatments, including the blank control. No survivors (100% mortality) were found when winter wheat seedlings germinated from untreated seeds were challenged with C. psychromobidus. A significant improvement in viability of winter wheat seedlings was observed when the seedlings were previously treated with T. atroviride biotype 603 (70% mortality) or CHS 861(50% mortality). No difference was observed between uninfected plant treated with CHS 861 (39% mortality) and the blank control (39% mortality).

       Examination of the sheath of T. atroviride-treated winter wheat grown in snow mold contaminated soils revealed a dramatic reduction in the number of sclerotia formed by C. psychromobidus. T. atroviride seemed to forge a symbiotic relationship with the roots of winter wheat plants; T. atroviride mycelia, chlamydospores and chlamydospore primordia-like structures were observed in the root cells of winter wheat with no adverse effects on the plants.

       Efficacy trials conducted on commercial golf courses in Wisconsin and Illinois showed promise-the severity of snow mold infestation was significantly reduced in plots treated with T. atroviride.

Usage of T. atroviride as a biofungicide

       To facilitate the ease of application and increase effectiveness in disease control, several formulation have been developed.

a)    Wettable formulation
b)    Flowable formulation
c)    Seed coating formulation

Both of the wettable and flowable formulations can be applied as a soil treatment, root dip, or foliar spray. They can also be used in potting mix.

Effects of Trichoderma atroviride on Human Health and Environmental safety

       Trichoderma app. is ubiquitous. It is commonly found in the soils of Alaska. It is but a part of a food chain in nature: it feeds on plant pathogenic fungi and is in turn being fed on by other microorganisms.

       The maximum growth temperature of T. atroviride is 33C. The characteristics of becoming acquiescent at temperatures beyond the maximum temperature make it benign to humans, other animals, and birds.

       Trichoderma atroviride does not have any adverse effects on human. In more than ten years and handled by large numbers of people both in my and my collaborator's labs, no one who came in contract with T. atroviride has shown any sign of hypersensitivity or discomfort. A comprehensive literature search in Medline, which contain nearly nine million records, yielded no positive finds.

       No record of adverse effects of T. atroviride on humans, animals, avian, fish, or aquatic invertebrates was found in a search conducted of the Biological Science Set of Cambridge Scientific Abstracts (2,200,000 records), Aquatic Sciences and Fisheries Abstract (600,000 records) and Biosis (10,400,000 records).

   Copyright 2003 AmPac Biotech.  All rights reserved.