Biocontrol of Fusarium wilt of cucumber with Trichoderma longibrachiatum NGJ167 (Rifai)

Aim: This research investigated the use of Trichoderma Results: The control plants had higher incidence and severity of F. oxysporum than the T. longibrachiatum -treated plants. The T. longibrachiatum NGJ167-inoculated Marketmoor had higher fruit weight value of 200g in the screenhouse when compared with the control which had a fruit weight value of 133.33 g. On the field, T. longibrachiatum -treated Marketmoor produced the highest fruit weight of 220 g while the control had a mean weight of 120.6 g. Results also revealed that T. longibrachiatum DNAs were absent in the inoculated cucumber fruits. Conclusion: The use of T. longibrachiatum NGJ167 as a biocontrol agent indicates its potentials in improving plant health in agriculture. The absence of T. longibrachiatum NGJ167 in the treated cucumber indicated that the consumption of such fruits will have no adverse effect on consumers’ health.


INTRODUCTION
Cucumber (Cucumis sativus L.) is a popular vegetable crop of the family Cucurbitaceae comprising 70 genera and 750 species [1]. The nutritional composition of cucumber fruit per 100 g edible portion is 3% carbohydrate, 1% protein, 0.5% total fat and 1% dietary fibre [2]. However, cucumber is susceptible to many pathogens and pests [3]. Fusarium oxysporum is one of the most important phytopathogens causing Fusarium wilt disease in more than a hundred species of plants [4]. Cucumber Fusarium wilt disease is one of the most serious fungal diseases in cucumber production in the world [5,6]. Generally, it caused cucumber yield losses of ~10% to 30% and poor quality products resulting in severe economic losses [7]. Cucumber Fusarium wilt disease may occur at all growth periods of the cucumber plant [8]. The pathogen can survive as durable spores for many years with or without plant debris in soil, and it retains the ability to infect cucumber plants causing pre-or post-emergence damping-off, vascular discoloration of roots and stems, and eventually the entire plants wilt or die. The disease management of Fusarium wilt usually consists of soil fumigation, seed treatment, use of disease resistant varieties and biocontrol bacteria to reduce infection and disease severity [8,9]. Some antagonists show potential to suppress this disease, such as mycorrhizal fungi and Trichoderma [10], Penicillium [11], Streptomyces [12] and Bacillus [13].
Trichoderma spp. are among the most frequently isolated soil fungi and present in plant root ecosystems [14]. These fungi are opportunistic, avirulent plant symbionts, and function as parasites and antagonists of many phytopathogenic fungi, thus protecting plants from diseases. Depending upon the strain, the use of Trichoderma in agriculture can provide numerous advantages: (i) colonization of the rhizosphere by the fungus (''rhizosphere competence'') allowing rapid establishment within the stable microbial communities in the rhizosphere (ii) control of pathogenic and competitive/deleterious microflora by using a variety of mechanisms (iii) improvement of the plant health and (iv) stimulation of root growth [14]. Chemical control agents are implicated in ecological, environmental and human health problems and pathogens can develop resistance to them. However, the use of biological control agents such as T. longibrachiatum are non-toxic to human health and once colonized by the plant roots, may last for several years. Therefore, the objectives of this work are (1) to determine the effectiveness of T. longibrachiatum NGJ167 as a biocontrol agent of F. oxysporum in cucumber both in the screenhouse and on the field and (2) to detect the presence of T. longibrachiatum NGJ167 genes in the cucumber fruits using Polymerase chain reaction.

Sources of Fungal Isolates
Trichoderma longibrachiatum NGJ167 (Rifai) was obtained from the Pathology Laboratory of International Institute of Tropical Agriculture (IITA), Ibadan while Fusarium oxysporum was isolated from cucumber plant rhizosphere by weighing 10g of of soil sample into a conical flask containing 90 mls of sterile distilled water. The suspension was shaken vigorously and serially diluted. Aliquots of 1 ml each from the serial dilutions were placed into sterile Petri-dishes and molten PDA was poured on them. The plates were swirled to obtain homogenous mixtures on the inocula and PDA. Plates were incubated at 25 º C for 72 hrs. The plates were observed for the appearance of Fusarium in the mixed cultures. Fusarium oxysporum was identified in the laboratory using cultural, microscopic and molecular methods. The two organisms were maintained in the laboratory by periodic transfer onto PDA slant and kept in the refrigerator at 4 º C until when required.

Pathogenicity test for Fusarium oxysporum
Pathogenicity test was confirmed using dipping method [15]. Conidial suspension of F. oxysporum was harvested by flooding the culture plate with sterile distilled water and gently scraped with spatula. Thereafter, the conidia were filtered through three layers of cheese-cloth and adjusted to a final concentration of 10 6 microconidia/ml using heamocytometer. Cucumber leaves were dipped in spore suspensions for 5 minutes while leaves dipped in sterile distilled water served as the control. Both inoculated and control treatments were incubated for 7 days at room temperature for disease development. To fulfil Koch's postulate, reisolation of the pathogenic fungus was done and compared with the original isolate.

PCR-based
Assay for the Identification of F. oxysporum Genomic DNA was extracted from 72 h broth culture of F. oxysporum using Zymo Research (ZR) fungal/bacterial DNA MiniPrep TM kit. The total DNA extracted from F. oxysporum was used as template in polymerase chain reaction (PCR) using primers, Forward (5`-ATG GGT AAG GAA GAC AAG AC -3`) and Reverse (5`-GGA GGT ACC AGT GAT CAT GTT -3`), which have been designed to amplify approximately 700bp from the translational elongation factor (TEF) gene region of F. oxysporum [16].

Dual Culture of Fusarium oxysporum and Trichoderma longibrachiatum
The identified F. oxysporum was co-cultured on duplicate plates of PDA with the T. longibrachiatum NGJ167 (Rifai) obtained from IITA, Ibadan in order to determine the biocontrol ability of T. longibrachiatum. This was achieved by preparing conidial suspension of T. longibrachiatum NGJ167 and 1.0 ml (10 6 microconidia/ml) of this suspension was put into a sterile Petri-dish. Molten PDA (45 º C) was poured into the plate. The plate was swirled to ensure the mixing of T. longibrachiatum NGJ167 and the agar. After the agar had solidified, 1.0 ml (10 6 microconidia/ml) of F. oxysporum conidial suspension was inoculated into the plate and a sterile glass spreader was used to spread the pathogen on the surface of the plate.

Soil Treatment and Planting Operations
The experiment was conducted both on the field and in the screenhouse. The screenhouse experiment was conducted between July, 2012 and September, 2012. The top soil for planting was collected from a depth of 3-5cm with a disinfected hand trowel. The soil was sterilized at 100ºC for 3 h to eliminate pathogenic microorganisms. Mycelial plugs from actively growing Trichoderma longibrachiatum NGJ167 on Potato dextrose agar (PDA) were inoculated into the soil and treatments were carried out as follows: (i) seedlings and T. longibrachiatum in the same 3 cm deep hole (T1) (ii) seedlings in 3 cm deep hole with T. longibrachiatum placed 3 cm below the seedlings (T2) (iii) seedlings in 3 cm deep hole with T. longibrachiatum placed on one side of the seedlings (T3) (iv) seedlings in 3 cm deep hole with T. longibrachiatum placed 3 cm on both sides of the seedlings (T4) (v) seedlings in 3 cm deep hole with agar plug of PDA without T. longibrachiatum (control). In the screenhouse, two weeks after the treatment, 100 ml spore suspension (10 8 cfuml -1 ) from 8-day old F. oxysporum culture was used to inoculate the soil in which the treated seedlings and the control were grown. However, F. oxysporum was not inoculated into the soil on the field; natural infection was allowed to set in. Plants were watered regularly. The first data was taken two weeks after pathogen inoculation and afterwards data were at 2 weeks interval on fungal incidence, severity and fruit weight.

Field Experiment
The field experiment was conducted at the vegetable field of National Horticultural Research Institute, Ibadan, Nigeria (Latitude 70 54'N, and Longitude 30 54'E, 213 meters above the sea level) between October and December, 2013. Ibadan is in the rain forest-savanna transition ecosystem of South-West Nigeria. Randomized complete block design (RCBD) was used for the field experiment. The randomization was generated on the computer using random table generator. There were four replicates and each replicate represented a block. In each block there were 5 treatments as stated above and each treatment represented a plot. For the two varieties, each plot was separated from the adjacent one by a distance of 1 m while plants within each plot were spaced 50 cm x 75 cm. Similar data taken in the screenhouse were also taken on the field.

Measurement of Disease Incidence
Disease incidence was estimated by counting the number of symptomatic plants and expressing it as a percentage of the total plants sampled. Recording of disease incidence was carried out 4 weeks after transplanting (WAT). The incidence of F. oxysporum wilt disease was recorded by visual symptom observation such as necrosis, wilting and plant death characteristic of the infection. The visible symptoms of the disease were critically observed and the infected plants were identified according to Givord et al. [17]. Disease incidence was estimated by counting the number of symptomatic plants and expressed as a percentage of the total plants sampled.

Measurement of Disease Severity
Disease severity was assessed using a 1-5 scoring scale, where (1= no visible symptoms; 2= symptoms on less than 25% of the plant; 3= symptoms cover 50% leaf area; 4= symptoms on entire leaf area and 5= stunting, deformation and death of plant [18]. This was carried out at 4 weeks after planting (WAT).

Measurement of Fruit Weight
The effect of the different treatments on fruit weight was determined by using a sensitive weighing balance (Mettler Toledo) to determine the weight of fruits in grams (g).

Evaluation of Cucumber for the Presence of Trichoderma longibrachiatum (Rifai) Genes
The extraction of T. longibrachiatum DNAs from cucumber fruits was achieved by using the cetyltrimethylammonium bromide (CTAB) procedure as described by Abarshi et al. [19]. The total DNAs extracted from the fruits were used as templates in PCR using the following universal PCR primers: Forward (5`-TCC GTA GGT GAA CCT GCG G -3`) and Reverse (TCC TCC GCT TAT TGA TAT GC -3`) [20] for the amplification of the internal transcribed spacer (ITS1 and ITS2) regions of T. longibrachiatum [20].

Statistical Analyses
Data obtained were subjected to Analysis of Variance (ANOVA) using the Statistical Package for Social Scientists (SPSS) version 16.0 and means were compared using Duncan's Multiple Range Test at P<0.05.

Characteristics of Isolated Fusarium oxysporum
Mixed fungal culture was obtained during the isolation of F. oxyporum. The fungi that were present in the mixed culture include Fusarium oxysporum, Aspergillus niger, A. flavus, Penicillium chrysogenum, P. notatum and Rhizopus spp. The 5-day old pure culture of F. oxysporum obtained from the mixed fungal culture revealed that the colonies had pink colour with cottony surface texture. The reverse side of the agar had pink pigmentation. The colony margin was smooth with semi raised elevation (Fig. 1). The morphological characteristics revealed that the hyphae were septate. They had conidiophores which were not well differentiated from the hyphae. Both macro-and micro-conidia were present. They had brown chlamydospores which were solitary (Fig. 2). The isolate was identified as Fusarium oxysporum after further identification using PCR.

Pathogenicity Test
When the isolated F. oxysporum was inoculated into cucumber leaves, necrotic lesions were observed indicating the presence of F. oxysporum in the plant. However, the cucumber leaves inoculated with sterile distilled water did not show any symptom.

PCR-based Assay for the Identification of F. oxysporum
The gel electrophoresis result of the F. oxysporum Translation Elongation Factor (TEF) gene showed that the gene responsible for Fusarium wilt disease in plants was extracted from the isolate identified as F. oxysporum. The PCR amplification product of the TEF gene gave a product size of 650 bp (Fig. 3).

Dual culture of Fusarium oxysporum and Trichoderma longibrachiatum
The antagonistic T. longibrachiatum suspension was able to grow faster than Fusarium oxysporum and made contact with colonies of F. oxysporum after 48 h of co-culturing.
Trichoderma longibrachiatum overgrew colonies of the pathogen and formed green clusters on F. oxysporum (Fig. 4).

Incidence of Fusarium oxysporum in Cucumber Grown in the Screenhouse
The control of the Ashley cucumber had the highest incidence of 68.33% while T2 had a value of 53.37% and the lowest incidence in Ashley was recorded in T1 with a value of 38.58%. However, 63% was the highest incidence recorded for the Marketmoor cucumber and this value was from the control plant. In the T.
longibrachiatum-treated Marketmoor cucumber, T2 had an incidence value of 44.29% while it was less in T4 with a value of 25% (Fig.  5).

Severity of Fusarium oxysporum in Cucumber Grown in the Screenhouse
The control of Ashley had a severity score of 4 indicating that the plants were severely infected with F. oxysporum while the T. longibrachiatumtreated plants were moderately infected with severity score of 2. With the exception of T1 which had a score of 3. The severity of infection of F. oxysporum on the control of Marketmoor was high with a score of 4.5 which indicated that the plants had combinations of leaf yellowing, necrosis and wilting. The T. longibrachiatumtreated Marketmoor had severity scores which varied from 2.5 to 3 with T1 and T4 having the least score (Fig. 6).

Dual culture of Fusarium oxysporum and Trichoderma longibrachiatum
The antagonistic T. longibrachiatum suspension was able to grow faster than Fusarium oxysporum and made contact with colonies of F. oxysporum after 48 h of co-culturing.
Trichoderma longibrachiatum overgrew colonies of the pathogen and formed green clusters on F. oxysporum (Fig. 4).

Incidence of Fusarium oxysporum in Cucumber Grown in the Screenhouse
The control of the Ashley cucumber had the highest incidence of 68.33% while T2 had a value of 53.37% and the lowest incidence in Ashley was recorded in T1 with a value of 38.58%. However, 63% was the highest incidence recorded for the Marketmoor cucumber and this value was from the control plant. In the T.
longibrachiatum-treated Marketmoor cucumber, T2 had an incidence value of 44.29% while it was less in T4 with a value of 25% (Fig.  5).

Severity of Fusarium oxysporum in Cucumber Grown in the Screenhouse
The control of Ashley had a severity score of 4 indicating that the plants were severely infected with F. oxysporum while the T. longibrachiatumtreated plants were moderately infected with severity score of 2. With the exception of T1 which had a score of 3. The severity of infection of F. oxysporum on the control of Marketmoor was high with a score of 4.5 which indicated that the plants had combinations of leaf yellowing, necrosis and wilting. The T. longibrachiatumtreated Marketmoor had severity scores which varied from 2.5 to 3 with T1 and T4 having the least score (Fig. 6).

Dual culture of Fusarium oxysporum and Trichoderma longibrachiatum
The antagonistic T. longibrachiatum suspension was able to grow faster than Fusarium oxysporum and made contact with colonies of F. oxysporum after 48 h of co-culturing.
Trichoderma longibrachiatum overgrew colonies of the pathogen and formed green clusters on F. oxysporum (Fig. 4).

Incidence of Fusarium oxysporum in Cucumber Grown in the Screenhouse
The control of the Ashley cucumber had the highest incidence of 68.33% while T2 had a value of 53.37% and the lowest incidence in Ashley was recorded in T1 with a value of 38.58%. However, 63% was the highest incidence recorded for the Marketmoor cucumber and this value was from the control plant. In the T.
longibrachiatum-treated Marketmoor cucumber, T2 had an incidence value of 44.29% while it was less in T4 with a value of 25% (Fig.  5).

Severity of Fusarium oxysporum in Cucumber Grown in the Screenhouse
The control of Ashley had a severity score of 4 indicating that the plants were severely infected with F. oxysporum while the T. longibrachiatumtreated plants were moderately infected with severity score of 2. With the exception of T1 which had a score of 3. The severity of infection of F. oxysporum on the control of Marketmoor was high with a score of 4.5 which indicated that the plants had combinations of leaf yellowing, necrosis and wilting. The T. longibrachiatumtreated Marketmoor had severity scores which varied from 2.5 to 3 with T1 and T4 having the least score (Fig. 6).

Incidence of Fusarium oxysporum Infected Cucumber Grown on the Field
The incidence of F. oxysporum in the control of Ashley cucumber was 63% and this was highest incidence. Of all the T. longibrachiatum-treated Ashley cucumber, T2 had an incidence of 50% while T4 had an incidence of 40.95% (Fig. 7).

Incidence of Fusarium oxysporum Infected Cucumber Grown on the Field
The incidence of F. oxysporum in the control of Ashley cucumber was 63% and this was highest incidence. Of all the T. longibrachiatum-treated Ashley cucumber, T2 had an incidence of 50% while T4 had an incidence of 40.95% (Fig. 7). The result obtained from Marketmoor cucumber revealed that 53.3% of the control plants were susceptible to Fusarium wilt infection.

Incidence of Fusarium oxysporum Infected Cucumber Grown on the Field
The incidence of F. oxysporum in the control of Ashley cucumber was 63% and this was highest incidence. Of all the T. longibrachiatum-treated Ashley cucumber, T2 had an incidence of 50% while T4 had an incidence of 40.95% (Fig. 7). The result obtained from Marketmoor cucumber revealed that 53.3% of the control plants were susceptible to Fusarium wilt infection.

Severity of Fusarium oxysporum Infected Cucumber Grown on the Field
The severity of F. oxysporum in the control of Ashley cucumber was 3.33 which was the highest while the least score was 1.33 by T4. In Marketmoor cucumber, the control had a severity score of 3. Apart from T2 which had a severity score of 2, the other T. longibrachiatum-treated plants had severity score of 2.33 (Fig. 8).

Effect of Fusarium oxysporum and Trichoderma longibrachiatum on Fruit Weight of Cucumber Varieties
The control of Ashley cucumber variety grown in the screenhouse had mean fruit weight value of 75 g which was significantly different (P < 0.05) from the mean values of the T. longibrachiatumtreated plants. Treatments T1 and T2 had the highest significant values of 201 g and 190 g respectively followed by T4 and then T3. Similarly, the control of Marketmoor cucumber grown in the screenhouse had the least significant fruit weight value of 133.33 g while the treated plants had higher values with T2 having the highest value of 200 g ( Table 1).
The fruit weights of Ashley cucumber grown on the field showed that the control had the least fruit weight of 76.67 g. The fruit weights of the T. longibrachiatum-treated Ashley were not significantly different at (P < 0.05) with mean values ranging between 120 g and 128.33 g, except T2, which had an average fruit weight of 106.67 g. The highest significant fruit weight of Marketmoor cucumber grown on the field was observed in T2 with an average weight of 220 g while the least weight of 120.6 g was observed in the control (Table 1).

Evaluation of Plants for the Presence of Trichoderma longibrachiatum (Rifai) Genes
The result of the agarose gel electrophoresis indicated that T. longibrachiatum DNAs were not present in the plant materials (Fig. 9). This implies that T. longibrachiatum-treated crops are safe for consumption.

DISCUSSION
In this study, Fusarium oxysporum was isolated from the rhizosphere of cucumber plant. The presence of this pathogen in the soil can be   linked to the fact that plant rhizospheres are rich in photosynthates which are utilized by microorganisms. There are several studies on the isolation of F. oxysporum from agricultural soils. Leslie and Summerell [21] reported that the genus Fusarium is a ubiquitous soil saprophyte and has been isolated from debris and roots, stems and seeds of a wide variety of plants.
The pathogenicity tests carried out between F. oxysporum and cucumber revealed that Koch's postulate was established. This is because of the necrotic lesions and yellowing induced on the healthy plant after inoculation with F. oxysporum. Boughalleb and El Mahjoub [22] confirmed Koch's postulate by re-isolating F. oxysporum from watermelon seed and confirming it to be responsible for vascular wilt of the seedlings.
The genomic DNA extracted from the morphologically identified F. oxysporum was identified to be F. oxysporum using PCR. The result obtained proved that the primer pair allowed a fast, reliable and specific identification of Fusarium oxysporum isolate and could be suitable for early diagnosis of Fusarium wilt of in soil. The use of species specific primers for the identification of Fusarium species has been described in many literatures [23,24].  When T. longibrachiatum cell suspension was grown in dual culture with F. oxysporum, the growth of the pathogen was inhibited by T. longibrachiatum. This could be attributed to the antagonistic ability of T. longibrachiatum, thereby inhibiting the growth of F. oxysporum. Also, competition between the two microorganisms for space and nutrients could be responsible for the growth inhibition of the pathogen. Viterbo et al.
[25] reported that T. harzianum IT-35 was able to control Fusarium species on various crops via competition for nutrients and rhizosphere colonization. The result on the safety of T. longibrachiatumtreated food showed that T. longibrachiatum was not present in the cucumber fruits obtained from the T. longibrachiatum-treated crops. The reason for this is that Trichoderma was not transported to the aerial parts of the plant. It was the hormones and chemicals that were communicated to the aerial parts. Benitez et al. [26] reported that the use of microorganisms that antagonize plant pathogens (biological control) is risk-free when it results in enhancement of resident antagonists.

CONCLUSION
The use of T. longibrachiatum as a biocontrol agent is promising in agricultural setting as it increases crop yield and improves plant health with no adverse effect on the environment and the consumers at large. The use of biocontrol agents should be a suitable alternative because of the adverse effects associated with chemical control agents.