Abstract: The aim of this two years research work is to recognize bio control agents and its antagonistic efficiency on Sclerotium rolfsii in vitro condition. Sclerotium rolfsii Sacc. is a causal agent of white foot and root rot disease in many plants. This fungi causing damage in tea nurseries of tea cultivated countries especially in Iran. Due to importance this disease in tea nurseries of Iran and impossibility using of chemical control against this damaging agent In this research, five species of Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum, Trichoderma longibrachiatum and Trichoderma parceramosum as antagonist fungi against Sclerotium rolfsii were collected and identified from tea nurseries and seedlings in Iran. Then efficiency of these bio control agent against Sclerotium rolfsii were investigated in vitro condition. Results showed that these species with different mechanism such as lysis of sclerotia, inhibited mycelial growth of Sclerotium rolfsii with volatile metabolites producing and parasitized the hyphal trends of disease agent were showed its antagonistic effects against causal agent of white foot and root rot in tea seedling. Trichoderma viride, Trichoderma harzianum and Trichoderma hamatum after 30 days destructed and lysis the sclerotia 98.5, 86.5 and 85%, respectively. Producing of volatile metabolites after 72 h reciprocal growth of Trichoderma viride, Trichoderma harzianum and Trichoderma longibrachiatum till 60.8, 54.8 and 54.4% prevented mycelium growth of Sclerotium rolfsii, respectively. All identified species caused parasitize (to hyphal contact method) pathogen fungal hypha that the first three species showed more parasite effect.
INTRODUCTION
Athelia rolfsii is telemorph of Sclerotium rolfsii Sacc. that belongs to Basidiomycete class and Basidiomycota phylum which over wintering in the form of sclerot and sometimes like mycelium (Alexopulus et al., 1996). This fungus is causal agent of damping off and also white foot and root rot disease of many important agronomical and horticultural plants. It is a polyphage fungus which has many hosts (Punja, 1985). This causal agent is one of the most important factors for damaging and losses in tea nurseries and seedlings in Iran that in some years even up to 50% loss of damping off and foot and root rot from tea nurseries is recorded (Okhovvat and Vakili, 1998). Infected seedlings in foot and root part were rotted, in such a way that in effected area the wood is became completely brown and plenty of mycelia were easily found. At the end of the season irregular and brown sclerot of fungus agent of disease were observed, that mostly considered as an over wintering factor (Khodaparast and Hedjaroud, 1996). S. rolfsii, was reported for the first time in Iran by Estuart (1953) on tea (Ershad, 1995). The studies have been stated that different species of Trichoderma have antagonistic effect against white foot and root rot disease (S. rolfsii). The researchers have been able to see the presence of hyphae, chlamydospores and conidia of Trichoderma koningii in the sclerot of S. rolfsii. They have reported that this bio control agent is caused destruction and lysis of sclerotia at disease agent fungus (Tsahouridou and Thanassoulopoulos, 2002a). Due to preference of biological control against foot and root rot disease and considering the references, the importance of S. rolfsii as causal agent in nurseries and seedlings, the research were conducted to identify theses bio control agents and their efficiency. This research work is the first identification, introduction and efficiency record of these bio control agents regarding foot and root rot and damping off disease in tea seedlings reported in the world.
MATERIALS AND METHODS
This research was conducted for two years in 2003-2004, with financial support of tea research institute of Iran (Lahijan, Guilan Province).
Extraction of Sclerotium rolfsii: In this research doubtful disease samples from nurseries of tea research stations and private gardens were collected during different seasons. After sterilization (one or two minutes deep in HgCl2) cultured the samples on PDA (Potato Dextrose Agar) media. Then the samples were identified with the help of key book (Barnett and Hunter, 1972) and purified by Hyphal tip method.
Extraction of antagonistic fungi: To extract antagonistic fungi 100 samples of soil from infected areas in tea nurseries and seedlings were collected. The samples were kept in pot for one week and moistured every alternate day to activate bio control agent. Twenty gram of soil from every pot were dissolved in 500 mL of 2 ppt citric acid till uniform suspension. Five milliliter of suspension were poured in a plate contain 15 mL of 50 °C agar and shake slowly till it became uniform. Four plates from each sample were prepared out of each plate five rings (15-20 mm) in diameter were taken, cultured on Davet media and kept in 25-27 °C incubator. After antagonist growth on Davet media, they were purified by single spore method and identified by key book (Rifai, 1969).
Investigation on the efficiency of Trichoderma spp. against S. rolfsii by hyphal contact: From each species of antagonist anted S. rolfsii (2 mm in diameter) disk were collected and kept on each side of sterilized slide covered with thin layer of PDA in three replications. Slides were kept inside plates, incubated in 25-27 °C. The contact effects of Trichoderma spp. growth against S. rolfsii were investigated by microscope.
Investigation on the efficiency of Trichoderma spp. volatile metabolites on mycelium growth of S. rolfsii: After recognition of Trichoderma (5 species) young and purified culture of each antagonist, a S. rolfsii were prepared in plates. Every plates of S. rolfsii were inverted against each Trichoderma species (15 plates from five species of Trichoderma and each species with three replications). One treatment selected as control, inverted against PDA. media. All plates were tightened with parafilm and kept for 72 h in (25-27 °C) incubator. Suppression percentage of S. rolfsii mycelium growth due to volatile metabolites of Trichoderma was calculated by the following formula:
Lysis effect on sclerot of S. rolfsii by Trichoderma species: In this study, plates containing fresh culture of S. rolfsii were kept in 25-27 °C incubator for 15 days. After sclerot formation, they were collected from each plate in culture room near fire with the help of sterilized forceps and incubated in 4 °C. Then all species of Trichoderma were cultured in PDA media; four plates from each species were purified. When mycelium growth of each antagonist reached up to 9 cm, they were taken out from incubator and 20 sclerots of S. rolfsii were kept over the colony of Trichoderma in 25-27 °C for one month. Then after lysis percentage of sclerotia were evaluated with the help of binocular.
RESULTS
Sclerotium rolfsii were recognized as causal agent of white foot
and root rot and damping off by taking sample from nurseries and seedlings
of tea research stations as well as farmers garden and cultured on PDA
media (Fig. 1a-c).
| Fig. 1: | White foot and root rot (S. rolfsii): (a) Infected tea nursery with sclerot of causal agent (b) Severely infected seedling of tea and (c) Purified S. rolfsii with sclerot in plate at in vitro condition |
Collected soil samples from nurseries with less loss percentage were cultured on Davet media, five species of Trichoderma were recognized with the help of key book (Rifai, 1969), reconfirmed by Iranian Research Institute of Plant Protection and Dr. Rouhani from Bouali University of Hamadan (Iran). Identified antagonist species were: Trichoderma viride, T. harzianum, T. hamatum, T. longibrachiatum and T. parceramosum. After identification of Trichoderma genus as antagonist species and reconfirmation by qualified institute in Iran, efficiency of these species with recognized mechanisms, such as hyphal contact, colonization ability, volatile metabolites and lysis of sclerot (Dennis and Webester, 1971a-c) were investigated an devaluated with three replications for two years in vitro condition. The efficiency results of Trichoderma species against white foot and root rot disease and damping off on tea in vitro explained on Table 1. After registration the records every year, annual analysis and combined analysis of data with the help of SPSS and data contrast of mean with the help of Danken test in 5% probability were calculated. The antagonist efficiency results against causal agents showed that extracted identified antagonist characters were different according to the experimented mechanisms (Table 1).
In hyperparasitism activity by hyphal contact method, T. viride, T. hamatum and T. harzianum had parasitism ability but the first two antagonists were more powerful where as the remaining two antagonists could not parasitized the causal agent (Fig. 2a-f).
In dual culture experiment, T. viride showed maximum colonization ability against S. rolfsii and produced spores after 72 h; could completely colonized the mycelium of S. rolfsii, although T. harzianum and T. hamatum were also able to colonized the causal agent but they had less ability in comparing to T. viride and produced spores later (Fig. 3a-e).
In the inhibitory ability experiment, volatile metabolites extract from
T. viride were most effective on mycelium growth of S. rolfsii
and its suppressive percentage after data contrasts of mean were in maximum
statistical group (a), then after T. harzianum and T. hamatum
were in lower group (b) and finally the other two antagonists were in
the lowest group (c) (Table 1).
| Table 1: | Efficiency results of Trichoderma sp. against S. rolfsii causal agent of white foot and root rot and damping off at tea seedling in vitro condition |
| ++a: High suppressive effect no overlapping of antagonist with S. rolfsii, +a: Medium suppressive effects no overlapping of antagonist with S. rolfsii, -a: No suppressive effect and overlapping of antagonist with S. rolfsii, ++b: High colonization ability of antagonist no overlapping, +b: Medium colonization ability of antagonist no overlapping, -b: No colonization ability of antagonist with overlapping, *, **: Means with the same letter(s) in each column are not significantly different at 5% level of probability | |
| Fig. 2: | The efficiency of Trichoderma spp. against S. rolfsii by hyphal contact: (a) T. viride, (b) T. hamatum, (c) T. harzianum, (d) T. longibrachiatum, (e) T. parceramosum and (f) Control |
| Fig. 3: | The efficiency of Trichoderma spp. against S. rolfsii by dual culture: (a) T. viride (b) T. harzianum, (c) T. hamatum, (d) T. longibrachiatum and (e) T. parceramosum |
T. viride, T. harzianum and T. hamatum species were most effective to destruct and lysis the sclerot. Within 30 days these three species destructed the sclerots of S. rolfsii 98.5, 86.5 and 85%, respectively. Two other species also could destruct the sclerots but they were in the lowest group (c) statistically (Table 1).
DISCUSSION
According to the experimented results, species of Trichoderma with different mechanisms have antagonistic efficiency against S. rolfsii, which reported by other researchers on different hosts (Papavizas, 1985; Chet, 1987; Harman et al., 1980; Elad et al., 1982). Sclerot of the fungi which remains over winter plays the most important role in its life cycle (Tsahouridou and Thanassoulopoulos, 2002a; Santos and Bettiol, 2003). Recorded results of this survey showed that in different species of Trichoderma, the best efficiency against damping off and white foot and root rot disease was lysis of sclerot, which was found that all five identified species could destruct and lysis the sclerot with average percentage of 82.6 by this mechanism, this naturally would be useful to control and decrease this causal agent at next year in vivo condition. According to conducted research work by other scientists and researchers, germination of over wintered sclerots is the main reason to start the disease with maximum population at the beginning of the season; although this fungus could over winter in the form of mycelium. Conducted research work in vitro condition showed that identified antagonist of some species which had efficiency effect against mycelium of causal agent could control by two mechanisms of volatile metabolites and hyperparasitism (Tsahouridou and Thanassoulopoulos, 2002b; Khodaparast and Hedjaroud, 1996; Zeppa et al., 1991). The results of this research work as a whole could explain that not only five species of Trichoderma extracted from nurseries and seedlings were identified but also these species with different mechanisms such as lysis of sclerots of causal agent S. rolfsii, prohibited the mycelium growth by volatile metabolites and hyperparasitism showed antagonistic effect and controlled damping off and white foot and root rot disease in tea seedlings. T. viridae, T. harzianum and T. hamatum species after 30 days could destruct and lysis the sclerots of causal agent by 98.5, 86.5 and 85%, respectively. Creating volatile metabolites after 72 h reciprocal growth of T. viridae, T. harzianum and T. longibrachiatum prevented mycelium growth of S. rolfsii by 60.8, 54.8 and 54.4%, respectively. All identified species parasitized mycelial growth by hyphal contact method of S. rolfsii that the first three species could parasitize better and were more effective. It was found necessary that, identified antagonist efficiency against S. rolfsii (the causal agent of white foot and root rot and damping off) should be investigated and examined in vivo condition also; the results of such survey would be reported by the authors in near future.