This study investigated the ability of Trichoderma spp. to control the Fusarium wilt of tomato, as well as the effect of these isolates on tomato plant growth in green house. Two native Trichoderma isolates [T. citrinoviride, (T33) and Trichoderma sp.anamorph of H. semiorbis, (T15)] significantly reduced tomato Fusarium wilt severity (19.77%) and increased all plant growth parameters (root length, 6.65 cm; shoot length,18.29; root fresh weight, 2.94g; shoot fresh weight, 28.66g; root dry weight, 0.46g; shoot dry weight,4.03g and no. of leaves, 24.33. The combination of both species of Trichoderma were more effective in controlling the Fusarium wilt disease compared to their application as alone.

Tomato (Lycopersicon esculentum Mill.) is one of the most important edible and nutritious vegetable crop grown all over the world [1]. Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. Lycopersici that is one of the economically most important disease in major tomato growing regions worldwide [2]. It is a highly destructive pathogen, causing 10 to 50% yield loss in many tomato production areas [3]. However, the widespread use of chemicals fungicides has been a subject of public concern and security due to their potentially harmful effects on environment and human health and their undesirable effect on non-target organisms [4,5].

The control of Fusarium wilt of tomato is very difficult because pathogen progress within the vascular tissues which limits the effectiveness of fungicides. Biological control of plant pathogens through antagonistic offer environmentally safe and cost effective alternative to chemicals [2]. Trichoderma has gained immense importance since last few decades due to its biological control ability against several deadly plant pathogens [6] for their ability to increase root growth and development, crop productivity, resistance to abiotic stresses, uptake and use of nutrients [7]. Several studies have been published on ability of Trichoderma in biological control of Fusarium wilt of tomato [8-10]. Therefore, the objective of the present study was to assess the ability of two Trichoderma spp. in decreasing Fusarium wilt of tomato under in vivo conditions in Yemen.

Origin of Trichoderma isolates
Native isolates T. citrinoviride (T33) and Trichoderma sp.anamorph ofHypocrea semiorbis (T15)wereisolated from rhizosphere soil of corn in Yemen and selected for their high biocontrol activity against different soil-borne plant pathogens.

Origin of Fusarium oxysporum
F. oxysporum (AUMC 208) was collected from Mycological Center, Faculty of Science, Assiut University, Assiut, Egypt.

Sterilization of soil and seeds
Soil used through this experiment was clay soil, sand and peat moss at a ratio of 3:1:1 (w/w) then autoclaved for three successive days at 121°C for one hour/day. Pots of 15 cm ×10 cm diameter were surface sterilized with 1% sodium hypochlorite and were filled with autoclaved soil [11, 12]. Seeds of tomato were surface sterilized with 1% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water and air dried [13].

Preparation of F. oxysporum inocula
Fusarium oxysporum inocula was prepared for incula by growing on PDA and incubated for 7 days at 28°C. The suspension for inoculation was prepared by pouring 30 ml of sterile distilled water into each of petri dishes containing F. oxysporum culture and the conidia were dislodged with a cell spreader, filtered through cheesecloth and counted with a haemacytometer. It was set to 1x10⁶ conidia/ml [14].

Pathogenicity test
Pathogenicity test of F. oxysporum was carried out on tomato variety (Solanum lycopersicum L., cvs. ‘Rio grande, Faten) according to Jasnic ., et al. [15]. Pathogenicity test was tested by sowing of tomato seeds in artificially infected soil by F. oxysporum. Control plants were sown in soil with sterile distilled water. The severity of the Fusarium wilt was assessed from 2 weeks of inoculation up to 45 days. Disease incidence was determined as reported by Khanna., et al. [16]:

Percentage diseases incidence (DI %) =  n   × 100
                                                             N
Where:
n= number of plants showing wilts symptoms
N= Total number of plant sampled

Biological control of Fusarium wilt of tomato in greenhouse
The selected antagonists (T15 and T33) were tested for their ability to control the Fusarium wilt of tomato as well as their effect on tomato plant growth under greenhouse conditions. Each pot was taken with sterilized soil. Soil was infested with conidial suspension (30 ml) of F. oxysporum (1x10⁶ conidia/ml). Simultaneously after 2 days of pathogen inoculation, soils were inoculated with (30 ml) of Trichoderma isolates (1x10⁷ conidia/ml) and then pots were watered for 7 days before sowing. Ten tomato seeds were sown in each pot, three pot replicate for each treatment arranged in completed randomized design. Pots were kept under greenhouse condition till the end of the experiment [17]. The details of total 5 treatments as follow:
T (1): Control (without Fusarium and Trichoderma)
T (2): F. oxysporum
T (3): Trichoderma sp.anamorph of H. semiorbis+ F. oxysporum
T (4): T. citrinoviride + F. oxysporum
T (5): Trichoderma sp.anamorph of H. semiorbis +T. citrinoviride+ F. oxysporum

At the end of experiment, the plants removed from the pots, shoot and root length, fresh weight (FW), dry weight (DW) and number of leaves per plant were determined [18]. The percentage of disease severity was determined using the formula given by Song et al., [19]:

Severity ratings for Fusarium oxysporum inoculated plants were done using a scale of 1-6 as described by Marley and Hillocks [20]. Where:

1. no symptoms
2. chlorosis and wilting of the first branches
3. chlorosis and wilting of second and third branches
4. chlorosis above third, second and third branches may be lost
5. chlorosis and partial desiccation
6. complete death of plant

Statistical analysis
Statistical analysis was performed in order to determine the effect of treatments on observed parameters. Significance of treatments was tested by One-way Analysis of Variance (ANOVA) and Tukey’s test (at P< 0.05) was applied for the differences in mean values. All the statistical analyses were completed using Graphpad 6.01 Statistics.

The tomato plants were inoculated with F. oxysporum to test the capability of the fungusproducing disease symptoms in tomato in vivo. In the present study theinoculated plant showed wiltingsymptoms and percentage of diseases incidence was (62.64%). The result is supported by the findingsof Begum [21] who observed that F. oxysporum f. sp. lycopersici was able to produce wilting symptoms in tomato plants.

The results of severity of F. oxysporum on tomato gave the highest percentage of disease severity (67.85%) (Figure, 1) and reduced all plant growth parameters (Table, 1). The negative effects of F. oxysporum result from disruption to plant growth by the blocking of xylem vessels, causing leaf senescence and reduced photosynthesis [22].

It was revealed from the results, Trichoderma isolates varied in their effect on tomato plants and ability to reduce the effect of F. oxysporum when subsequently applied in the pot experiment (Table, 2). Reduction of Fusarium wilt disease was observed by T. citrinoviride and Trichoderma sp.anamorph of H. semiorbis in comparison to control. Best disease control was achieved in treatment (5)demonstrating only 19.77% of disease severity followed by treatment (4) and treatment (3). These results are in agreement with previous studies of several workers [3,8, 23,24] they reported that isolates of Trichoderma showed promise for controlling Fusarium wilt and improving the growth and yield of tomato.

The reduced Fusarium wilt on tomato may have been due to reduced population density of the pathogen [25], parasitism, lysis of pathogenic fungi, competition for limiting factors in the rhizosphere mainly iron and carbon [26,27].

The results of Trichoderma treatments showed statistically significant effects of Trichoderma treatments on plant growth parameters (Table, 1). Root and shoot length, fresh weight dry weight and number of leaves per plant found that the maximum was recorded in the treatment [5]. These results agree with Ozbay and Newman [28] who reported that seedling treated with T. harzianum increases seedling emergence, number of true leaves, fresh and dry weights of shoot and root of tomato plants.

Sundaramoorthy and Balaskar [29] also observed that tomato plants treated with T. harzianum (ANR-1) stimulate plant height by 73.62 cm and increased the dry weight by 288.38 g in comparison to untreated control.

Furthermore, Harman [30] established that Trichoderma spp., are opportunistic plant colonizers that affect plant growth by promoting abundant and healthy plant roots, possibly via the production or control of plant hormones. The increased growth response caused by Trichoderma isolates may be through modification of the rooting system as Yedida., et al. [31] reported for T. harzianum inoculation which improved uptake of nutrients by the plants at a very early growth stage.

Richoderma viride and Trichoderma sp. Decreased disease incidence of Fusarium sp., and increased plant yield in tomato. Moreover, the combination of both Trichoderma spp. proved better than their application alone. Thus, the finding of present investigation holds a good promise in tomato wilt management. However, further studies on the effect of these treatments in field conditions need to be undertaken so that Trichoderma could be recommended as a biocontrol agent richoderma viride and Trichoderma sp. Decreased disease incidence of Fusarium sp., and increased plant yield in tomato. Moreover, the combination of both Trichoderma spp. Proved better than their application alone. Thus, the finding of present investigation holds a good promise in tomato wilt management. However, further studies on the effect of these treatments in field conditions need to be undertaken so that Trichoderma could be recommended as a biocontrol agent

T. citrinoviride (T33) and Trichoderma sp.anamorph of H. semiorbis (T15), decreased disease severity of F. oxysporum and increased plant growth parameters in tomato. Moreover, the combination of both Trichoderma spp. proved better than their application alone in vivo in biological control of Fusarium wilt of tomato.

Acknowledgement
We thank Faculty of Science, University of Sana’a for their support during this investigation.