Department of Agriculture, Payam e Noor University, P. O. Box 19395-4697, Tehran, Iran

© 2015, TextRoad Publication and Biological Sciences www.textroad.com

Effects of Trichoderma spp. in Bio-controlling Fusarium solani and F.

oxysporum of Cucumber (Cucumis sativus)

Mohammad Akrami

Department of Agriculture, Payam e Noor University, P. O. Box 19395-4697, Tehran, Iran

Received: November 2, 2014 Accepted: January 23, 2015

ABSTRACT

The experiment was conducted to assess the effect of three typical Trichoderma spp. (T. Harzianum or T1, T.

Asperellum or T2 and T. virens or T3) alone or in combination on pathogens of Fusarium solani and F.

oxysporum as agents of root rot in Cucumber (Cucumis sativus) field. A total twenty experimental cucumber fields was considered for study on basis of a completely randomized block design (with 4 replicates, 9 plants per). Plants were planted in Fusarium pre-infested soil and then treated with one or mix of Trichoderma spp.

(T1, T2, T3, T1+T2, T1+T3, T2+T3, T1+T2+T3) for detecting the best bio-control treatment against pathogens.

The F. solani-infested fields treated with mix trio of Trichoderma spp. (T1+T2+T3), showed the lowest external yellowing and dropping, root discoloration and disease incidence (10.50%, 6.50% and 11.25%, respectively). Better bio-control of F. solani and F. oxysprom was also done by different combination of antagonist funguses (80-84%). It concluded that the combination of Trichoderma spp. showed the more protective effects on cucumber root rots agents (Fusarium) and it can be considered as a protective strategy in fruiting plants for reduce the negative effects of infectious pathogens.

KEYWORDS: Fusarium, Trichoderma, Bio Control, Cucumber Field.

1. INTRODUCTION

Cucumber (Cucumis sativus L.) is a widely cultivated plant in the gourd family Cucurbitaceae. It is a creeping vine that bears cylindrical fruits that are used as culinary vegetables. Many different varieties are traded on the global market but there are three main varieties of cucumber: slicing, pickling, and burpless. Within these varieties, several different cultivars have emerged. The cucumber is originally from Southern Asia, but now grows on most continents.

Fusarium is an important and insidious disease which attacks plants and grains, especially in humid and semi-humid areas [1]. Worldwide distribution of cucumber production for 2010 was dominated by China (c.

70%), with the countries of Iran, Turkey, Russia and United States making up the other major producers respectively. Other significant production areas include Ukraine, Spain, Egypt, Japan and Indonesia. Several diseases are known to limit production of cucumber, of which Fusarium solani f. sp. Eumartii and Fusarium oxysporum f. sp. ciceris (Fusarium wilt) are the most important [2, 3]. Management of fusarium wilt has been primarily through development of resistant cultivars as part of an integrated management approach [4, 5].

Fusarium solani strain (F. solani (Mart.) Sacc. f. sp. Eumartii (C. Carpenter) W.C. Snyder & H.N.

Hans.) is a worldwide soil-borne fungus attacking a wide range of host plants including citrus [6], with a great overall impact on productivity. The disease caused by this fungus is characterized by wilted plants, yellowed leaves and root rot minimal or absent crop yield [7]. Studies showed that the most common citrus rootstocks are susceptible to dry root rot caused by F. solani, and their results tally with other field observations [8, 9].

F. oxysporum strains (F. oxysporum Schlechtend: Fr. f. sp. ciceris (Padwick) Matuo & K. Sato) are ubiquitous soil inhabitants that have the ability to exist as saprophytes, and degrade lignin [10] and complex carbohydrates [11] associated with soil debris; hence, named Fusarium wilt. They are also pervasive plant endophytes that can colonize plant roots [12] and may even protect plants or be the basis of disease suppression [13]. Although the predominant role of these fungi in native soils may be as harmless or even beneficial plant endophytes or soil saprophytes, many strains within the F. oxysporum complex are pathogenic to plants, especially in agricultural settings.

The biological control is the best alternative especially against soil borne pathogens. Biological control of pathogens, i.e., the total or partial destruction of pathogen populations by other organisms, occurs routinely in nature [14]. Among the various antagonists used for the management of plant diseases, Trichoderma spp. plays a vital role. Among the various isolates of Trichoderma, T. Asperellum, T. harzianum, T. virens, T. viride, and T.

hamatum are used against the management of various diseases of crop plants especially with soil borne pathogens.

These filamentous fungi are very common in nature, with high population densities in soil and plant litters [15].

Many studies have proved the potential of Trichoderma spp. as biological agents antagonistic to several plant pathogens [16-18]. Several biocontrol bacteria, including Pseudomonas spp., Serratia marcescens, Bacillus sp., Streptomyces sp. have been used to control Fusarium wilt diseases [19]. Moreover, use of Trichoderma spp. on

banana [20], arbuscular mycorrhiza (AM) on banana [21] and soil amendment of Lettuce on cucumber [5] have also been reported. Several workers have used combinations of microorganisms in biological control experiments [22].

The main objective of this research was to test the performance of Trichoderma spp. includes T.

Asperellum, T. harzianum and T. virens to suppress Fusarium root and stem rot of cucumber under greenhouse conditions.

2. MATERIALS AND METHODS

The present study was carried out in in Azerbaijan country and in part in East Azarbayjan Province, Iran.

F. solani (Mart.) Sacc. f. sp. Eumartii and Fusarium oxysporum f. sp. Lycopersici were isolated from cucumber roots according to method described by Nelson et al. [23]. Non-pathogenic fungal isolates (Trichoderma spp.), T. harzianum, T. Asperellum and T. virens were obtained from cucumber rhizosphere and field soil during the preliminary study according to methods described by Elad and Chet [24] and Harman (2006).

Both Fusarium spp. was subcultured on Potato Dextrose Agar (PDA) at 25± 1°C. 10 ml of each of Fusarium spp. Culture suspension (10⁷cfu/ ml) was added to soil of cucumber field. Trichoderma spp.: T.

harzianum, T. asperellum and T. viride were used alone or in combination; so that soil treatment by adding 10 mL from all or each of Trichoderma spp. for selected cucumber fields from a concentration of 10⁸cfu/ ml were added one week before Fusarium inoculation. Moreover, Trichoderma spores were pasted with 0.5% sugar to surface of seeds with a concentration of 3×10⁶, prior to implantation of cucumber seeds in soil infected to Fusarium.

Field soil infestation and treating

To perform this experiment, twenty soil fields of cucumber were infected by F. solani (Fs) and F.

oxysporum (Fo) and treated by T. harzianum (T1), T. Asperellum (T2), T. virens (T3), T1+T2, T1+T3, T2+T3 and T1+T2+T3 plus a cucumber field as control group.

Soil was artificially infested with pathogen fungi grown on moistened PDA at rate of 100 g m^-2 soil. Each treatment consisted of four replicate rows of 10 plants row^-1. Disease was monitored for 6-8 weeks and assayed as percentage of disease incidence (Table 1). Stem sections of wilted plants were surface-disinfested in 0.5% sodium hypochlorite and plated on PCNB medium to confirm the presence of the wilt pathogen. Stem sections of asymptomatic plants were also plated at the conclusion of the experiment to evaluate potential pathogen infection.

In the current study, plants were pre controlled one week prior to inoculation with the pathogen which appeared healthy and with no wilting or root rot symptoms. Wilt observed within 25 days of sowing into infected soil and when plants affected by pathogens that is determined via monitoring the most common symptoms such as drooping of the petioles, rachis and leaflets in the upper part of the plant, together with the pale green color of the foliage chlorosis of lower leaves, uprooted before completely dried.

Statistical analysis

All experiments were performed twice with four replicates per treatment and arranged in a randomized complete block design. Disease incidences (%) were analyzed using an Analysis of Variance (ANOVA) and grouped by Duncan test.

3. RESULTS

In the present study cucumber roots planted in soil infested by F. solani and F. oxysporum then treated with T1 (T. harzianum), T2 (T. Asperellum), T3 (T. virens), T1+T2, T1+T3, T2+T3, T1+T2+T3 for detecting the best bio control treatment (alone or in combination) against each of pathogen agents.

Cucumber cultivar inoculated with F. oxysporum f. sp. ciceri and R. solani, showed greater wilt incidence, chlorosis of leaves and induced vascular discoloration in roots. Soil administration with biocontrol agents adjusted the severity of wilt in roots, substantially (P<0.01). The F. solani-infested fields treated with mix trio of Trichoderma spp. (T1+T2+T3), showed the lowest external yellowing and dropping, root discoloration and disease incidence (10.50%, 6.50% and 11.25%, respectively). Trichoberma spp. bio control of cucumber fields exposed to pathogens significantly reduced Fusarium rot in field (Table 1). F. solani infested groups that treated with Trichoberma spp. caused to a significant decline in disease incidence, hence detected a well-control results.

The best bio control agents efficacy was related to cucumber fields controlled by ≥ 2 Trichoberma spp. (Figure 1).

The alone treatments showed a lower control especially in compared to Tr1+Tr2+Tr3 groups; however, cucumber roots rot well-controlled by T. harzianum (Fo+T1=81% and Fs+T1=82%) in compared with other T. asperellum (T2) and T. virens (T3). The lowest control was observed in fields infested to both Fusarium spp. treated with T.

virens (T3).

Table 1. Root discoloration and percentage of disease incidence in Cucumber fields treated with bio agents (Trichoderma harzianum, T. asperellum and T. virens) against Fusarium wilt caused by

Fusarium oxysporum and Fusarium solani.

Treatments^{1, 2} External yellowing and dropping Root discoloration % disease incidence %

Control 1.00g 0.25^g 0^h

Tr1 0.50g 0.00^g 0^h

Tr2 0.50g 0.25^g 0^h

Tr3 0.50g 0.25^g 0^h

Fo 88.75a 63.00^a 97.00^a

Fs 87.00 55.75^b 96.25^a

Fo + Tr1 13.75e 7.75^ef 18.50^dc

Fs + Tr1 16.50cd 7.50^ef 16.50^de

Fo + Tr2 17.25cd 9.50^df 18.75^de

Fs + Tr2 15.50ed 10.50^dc 15.25^fe

Fo + Tr3 20.25b 12.00^c 23.50^b

Fs + Tr3 18.50cb 11.00^dc 19.75^c

Fo + (Tr1+Tr2) 10.00f 7.75^ef 15.25^fe

Fs + (Tr1+Tr2) 11.25f 8.50^ef 16.00^fe

Fo + (Tr1+Tr3) 10.00f 8.75^de 13.75^fg

Fs + (Tr1+Tr3) 10.75f 8.50^def 13.50^fg

Fo + (Tr2+Tr3) 10.00f 8.75^de 13.50^fg

Fs + (Tr2+Tr3) 11.75f 8.00^de 15.75^fe

Fo + (Tr1+Tr2+Tr3) 9.50f 9.50^de 13.50^fg

Fs + (Tr1+Tr2+Tr3) 10.50f 6.25^f 11.25^g

SEM 0.698 0.760 0.806

P value *** *** ***

a-h, Values in the same row and variables with no common superscript differ significantly. ¹ Values represent the means of twelve observations per treatment and standard error of mean (SEM). ² Fs = F.sоlani, Fo = F.охyspоrum, Tr1 = T.harzianum, Tr2 = T.aspеrеllum, Tr3 = T.virеns; ^{3 *}: P<0.05, ^**: P<0.01, ^**: P<0.001.

Figure 1. percentage of d efficacy in chickpea fields treated with bio agents (Trichoderma spp.) against Fusarium wilt caused by Fusarium oxysporum and Fusarium solani. FS= Fusarium solani, FO= Fusarium oxysporum, T1= Trichoderma harzianum,

T2= Trichoderma Asperellum, T3= Trichoderma virens

DISCUSSION

The resistance was evident as a reduction in root discoloration in compared with infected control. As shown in Table 1, all treatments of Trichoderma either separately or in combination protected cucumber fields against F. oxysporum and Fusarium solani, a wilt and root rot factors, respectively. In the current study, when wilt observed in cucumber plants of field via monitoring the most common symptoms such as drooping of the petioles, rachis and leaflets in the upper part of the plant, together with the pale green color of the foliage chlorosis of lower leaves, uprooted before completely dried and we just evaluated the internal root discoloration for primary study of deep root rots (Table 1).

Haware et al. [25] reported that infected plants show no external root discoloration. However, internal discoloration may be seen extending up towards the stem. Internal discoloration is due to infection of the xylem tissues of the root and stem. Transverse sections of the infected root examined under the microscope show the presence of hyphae and spores of the fungus in the xylem. This is a diagnostic feature of Fusarium wilt [26]. In certain cucumber cultivars typical symptoms may not develop. Instead, there is a yellowing and drying of the lower leaves, and a stunting of the plant. Roots will show internal discoloration.

Based on above-mentioned information related to Fusarium wilt and rot of cucumber that was sourced from the National Diagnostic Protocol, it was revealed that disease incidence percentage in the presented study was significantly reduced by each one from controller agents compared with the infected controls (Fo and Fs, Figure 1). All treatments had a higher efficacy in inducing resistance; however, in Fusarium-infected and subsequently Trichoderma-treated cucumber fields, Disease incidences percentages were significantly reduced by biocontrol agents (Trichoderma spp.). Likewise, a better protection against disease incidence was observed when a combination of three T. spp. (T. harzianum, T. Asperellum and T. virens) were used for treatment of F. spp.- infected groups along with almost negligible differences compared with two or one of T. spp. (1 to 4% Disease incidences) with the exception of Fusarium-infected groups that separately treated with T. virens (T3). Our results are agreement with other findings [27-30].

The competitive ability of a non-pathogenic strain partly determines its capacity to establish in soil and in the plant rhizosphere and is probably involved in its capability to colonize the root surface demonstrated that different strains have different capacities to colonize heat treated soil [31].

In addition, saprophytic colonization of soil depends not only on the fungal strain but also on biotic and abiotic soil characteristics. Colonisation of the root surface and root tissues probably depends not only on the fungal strain but also on the plant species and plant cultivar. It was previously revealed that, within the Fusarium genus, F. oxysporum and Fusarium solani are without doubt the most economically important species [32].

In present study, the better efficacy was observed in treatments including T. harzianum (Figure 1).

Therefore, combination of Trichoderma spp. provided better disease control than alone isolates against Fusarium.

Trichoderma spp. is among the most-promising bio control fungi against many fungal plant pathogens. T.

harzianum has multiple mechanisms of action, including coparasitism via production of chitinases, β-1-3 glucanases and β-1-4 glucanases, antibiotics, competition, solubilisation of inorganic plant nutrients, induced resistance and inactivation of the pathogen's enzymes involved in the infection process [33, 34]. Different isolates of Trichoderma have different ability for fungal; their indirect effects may also vary. Therefore, one of the most interesting aspects of biology is the study of the mechanisms employed by biocontrol agents to affect disease control [35]. Possible mechanisms of antagonism employed by Trichoderma spp. includes nutrient and niche competitions, antibiosis by producing volatile components and non-volatile antibiotics [36,37] that is inhibitory against a range of soil borne fungi. Also, synergism between different forms of action modes occurs as the natural condition for the bio control of fungal pathogens [38]. It is widely known that environmental parameters such as abiotic (soil type, soil temperature, soil pH and water potential) and biotic (plant species and variety, and microbial activity of the soil) factors as well as other factors such as method and timing of applications may have influence on the biological control efficacy of Trichoderma isolates. Therefore, it is important that Trichoderma bio control potential in field should be further evaluated.

CONCLUSION

By using a combination of Trichoderma spp. such as T. harzianum, T. Asperellum and T. virens we could reduce disease incidence percentage of cucumber fields exposed to Fusarium pathogens such as F. solani and F.

oxysporum as agents of root and stem rot cucumber under greenhouse conditions. The presented method of bio controlling of pathogens can be considered as an applicable strategy in control measures against pathogens.

Acknowledgments

The authors are thankful to Dr. Doust Morad Zafari (Buali Sina University, Hamadan, Iran) for his attempts in conduction of laboratory assays and to A. Sh. Ibrahimov for his scientific assistance in terms of study.

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