In vitro Evaluation of Carbendazim 50% WP, Antagonists and Botanicals Against Fusarium oxysporum f. sp. psidii Associated with Rhizosphere Soil of Guava

INTRODUCTION

Wilt disease is a major limiting factor for the productivity and production of guava. The exact cause of disease is not fully understood and it is recognized as disease complex (Suhag, 1976) caused by more than one organism and possibilities not to be ruled out. All the strains of Fusarium exist saprophytically, but some are well known for inducing wilt (O'Donnell et al., 2000; Jurgenson et al., 2002; Schroers et al., 2005). Forma specials of Fusarium oxysporum causing wilt disease in Psidium guajava L. is designated as Fusarium oxysporum f. sp. psidii (Prasad et al., 1952; Gupta, 2010). Fusarium oxysporum f. sp. psidii mainly present in the rhizosphere soil and in vascular region of roots. Rhizosphere soils contain a wide variety of microbes viz. fungi, viruses, nematodes and bacteria, etc. Different type of interaction may takes place in the rhizosphere, which include effect of microflora on plant growth and different type of interactions like symbiosis, antagonism, synergism between the rhizosphere microfloras. Rhizosphere and Rhizoplane microbial population can influence the various aspect of plant growth and development like root morphology, root to shoot weight ratio, physiological process and crop yield, etc. Rhizosphere microorganisms, directly or indirectly effects the plant growth by making the nutrients available to the plants and by being antagonistic to phytopathogens. They influence the growth of plants and cause death in severe cases of infection. Edward (1960a, b) explained that Fusarium oxysporum f. sp. psidii penetrate either directly through the root piliferous layer of the guava seedlings or through openings caused by secondary roots. First external symptom of the guava wilt is the appearance of yellow colouration with slight curling of the leaves on terminal branches. Subsequently, browning, drooping and premature shedding of the leaves occur. Infected guava plants start showing sign of wilting right with onset of rainy season in August with maximum number dying in September and October (Das Gupta and Rai, 1947; Edward, 1960a; Suhag, 1976). As the wilt progresses, the fruits of affected branches remain under developed, hard and stony. Later on, the entire plant becomes defoliated and eventually dies, but hard, stony and dark brown and/or black fruits hang on the branches for sometime. Usually, fifteen days are requiring for the complete wilting, but some trees take even upto one year. The finer roots show black streaks, which become prominent on removing the bark (Das Gupta and Rai, 1947). The roots also show rotting at the basal region and the bark is easily detachable from the cortex. The cortical regions of the stem and root show distinct discolouration and damage. Light brown discolouration is also noticed in the vascular tissues (Chattopadhyay and Bhattacharjya, 1968). In general, above ten year old plants are more prone to the wilt incidence. Partial wilting is also common, when one side of the few branches wilt during the first year and in the next year full plant dies. Suhag (1976) observed that it is possible to regenerate even the worst affected trees by severe pruning followed by drenching with 0.2% either benlate or bavistin four times in a year and spray in twice with metasystox and zinc sulphate. Spread of the wilt could be checked by judicious amendments of N and Zn (Suhag and Khera, 1986). Pesticides are applied to plant and soil to control plant parasites and weed; they may also affect soil properties, micro-organisms and hosts (Altman and Campbell, 1977). Farmers are using very toxic pesticides, for the controlling of soil borne diseases as with caused by Fusarium sp. which are very hazardous to our flora and fauna. Biocontrol agents Trichoderma sp. and Streptomyces chibaensis have been suggested for the control of wilt pathogens (Dwivedi, 1992). Trichoderma sp. and Aspergillus niger were isolated from the rhizosphere of healthy guava plants and tested against the wilt pathogen in the laboratory (Singh et al., 2003). In addition to these control measures, use of many plant extracts and botanical fungicides have been found to be effective and gaining importance in crop production in the view of their selective action, low cost, environment friendly, long lasting effect, etc. to control of many plant diseases (Oros and Ujvary, 1999; Mamatha and Rai, 2004). The present study has been undertaken to evaluate the effects of some ecofriendly compounds against Fusarium oxysporum f. sp. psidii.

MATERIALS AND METHODS

Survey was conducted in 2005-2006 in five different places of Allahabad, district to collect the soil. One hundred soil samples were collected from the rhizosphere zone of wilted guava tree.

Isolation of Fusarium oxysporum f. sp. psidii was carried out with the help of serial dilution technique in PDA (Aneja, 2005). One milliliter of suspension (from rhizosphere soil) from dilution (10^-2) to (10^-5) was transferred in sterilized plates. Ten milliliter of PDA medium was poured in each plate and gently rotated to ensure uniform distribution of inoculums. After solidification, the plates were incubated at 28°C for 24 h in inverted position so that the condensed vapour may not hamper the growth of the fungus to be isolated. The pathogens were identified on the basis of colony characters, microscopic examination and Camera Lucida drawings. The culture of Fusarium oxysporum f. sp. psidii was purified from isolated dishes and maintained by periodic sub-culturing in PDA slants after every 15 days (Aneja, 2005).

Effect of carbendazim 50% WP on Fusarium oxysporum f. sp. psidii was done by poisoned food technique was followed at different ppm (Nene and Thapliyal, 2000). The principle involved in this technique is to supplement the nutrient medium with a toxic chemical and then allowing a test fungus to grow on such a medium and evaluated the effect of such chemical by measuring the radial growth of the fungus. The required dilutions of chemical (10,000, 1000, 100, 10 and 0 ppm) were prepared. Stock solution of 10,000 ppm, 0.2 g of active ingredient for respective chemical was added to 100 mL of sterilized PDA medium. Five treatments including control were applied in experiment and each treatment was replicated three times.

Bio-efficacy test of antagonists has been done against Fusarium oxysporum f. sp. psidii with the help of dual culture (Aneja, 2005). The effects of antagonists were studied by Zone of Inhibition technique (in vitro). The three antagonists were Aspergillus niger, Penicillium sp. and Trichoderma sp.

Formula to calculate I.O.C. % is:

Four treatments including control were applied in experiment and each treatment was replicated three times.

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Effect of neem leaf extract (Azadirachta indica), Datura leaf extract (Datura stramonium) and Lantana leaf extract (Lantana camera) at control, 5, 10 and 15% has been done against Fusarium oxysporum f. sp. psidii in three replications with the help of poisoned food technique (Nene and Thapliyal, 2000). Fifteen mililiter of extract was added in 85 mL of PDA medium for obtaining 15% concentration. Similarly 10 and 5% concentrations were prepared.

Inoculation of test organism: Measured quality of extract was added in sterilized Petri plates in three replications with treatments T₁, T₂ and T₃. No extract was added to Petri plate with treatment T₀ (control). Prepared medium was poured and spread uniformly in the Petri plates. After sometimes media was solidified, then with the help of sterilized cork borer, discs of 5 mm. diameter were cut from actively growing fungal cultures and then placed in centre of Petri plate under aseptic conditions containing the poisoned food. The inoculated Petri plates were incubated at 25°C and the radial growth of colony in mm. was measured after 2, 4 and 6 days of incubation.

In the present experiments, R.B.D. was applied. The analysis of variance technique was applied for drawing conclusions from the data. The calculated value was compared with tabulated value at 5% level of probability for the appropriate degree of freedom (Fischer and Yates, 1968).

RESULTS

Data presented in Table 1 reveals that different ppm of carbendazim 50% WP with pathogen Fusarium oxysporum f. sp. psidii was found to be significant. On 2nd, 4th and 6th days, radial growths of colony were not significantly different between treatments T₂, T₃ and T₄. The maximum radial growth of Fusarium oxysporum f. sp. psidii was found in T₀(control) followed by T₁ (10 ppm carbendazim 50% WP). The radial growth of Fusarium oxysporum f. sp. psidii was fully inhibited at 100 ppm (T₂), 1000 ppm (T₃) and 10,000 ppm (T₄) of carbendazim 50% WP.

Data presented in Table 2 reveals that in treatments viz., T₁ (Aspergillus niger + Fusarium oxysporum f. sp. psidii), T₂ (Penicillium sp. + Fusarium oxysporum f. sp. psidii) and T₃(Trichoderma sp. + Fusarium oxysporum f. sp. psidii), growth of Fusarium oxysporum f. sp. psidii was significantly inhibited as compared to T₀ (control). After 24 h, inhibited over control percentage of treatments T₁ (19.22) and T₃ (19.22) was similar followed by T₂(09.99). After 48 h, inhibition over control percentage of treatment T₃ (44.07) was maximum followed by T₁ (43.22) and T₂ (24.70). After 72 h, the maximum inhibition was observed in T₃ (61.91) followed by T₁ (61.12). The maximum inhibition over control percentage was in T₂ (49.22).

Table 1:	Effect of carbendazim 50% WP on the radial growth of Fusarium oxysporum f. sp. psidii at different ppm

S: Significant, SEM: Standard error mean

Table 2:	Effect of Aspergillus niger, Penicillium sp. and Trichoderma sp. on the growth of Fusarium oxysporum f. sp. psidii at different time intervals

A: Antagonist, P: Pathogen, Each value is mean of 3 replicates. I.O.C.: Inhibition over control. D1: After 24 h of incubation, D2: After 48 h of incubation and D3: After 72 h of incubation

Table 3:	Effect of neem leaf extract on the radial growth (mm) of Fusarium oxysporum f. sp. psidii at different concentrations

S: Significant. SEM: Standard error mean

Table 4:	Effect of Datura leaf extract on the radial growth (mm) of Fusarium oxysporum f. sp. psidii at different concentrations

S: Significant. SEM: Standard error mean

Table 5:	Effect of Lantana leaf extract on the radial growth (mm) of Fusarium oxysporum f. sp. psidii at different concentrations

S: Significant. SEM: Standard error mean

Data presented in Table 3 reveals that neem leaf extract at different concentrations was found to be significant to manage the growth of Fusarium oxysporum f. sp. psidii. The radial growths of colony on 2nd and 4th day were not significantly different from T₂ and T₃ and significantly different from T₀ and T₁. On 6th day, the radial growth was not significantly different between T₂ (07.83) and T₃ (07.33) as compared to T₁ (22.33). Data presented in Table 4 reveals that radial growth of colony on 2nd day were not significantly different between T₁(07.50), T₂ (07.30) and T₃ (05.83). On 4th day, the radial growth was not significantly different between T₂ (09.00) and T₃ (07.50). On 6th day, the radial growth was significantly different in T₀ (90.00), T₁ (39.33), T₂ (26.33) and T₃(18.67). Data presented in Table 5 reveals that radial growth of colony on 2nd day were significantly different in T₀ (31.67), T₁ (07.00), T₂ (06.33) and T₃(05.67). On 4th day, the radial growth was significantly different in T₀ (66.33), T₁ (12.33), T₂ (09.00) and T₃(07.33). On 6th day, the radial growth was significantly different in T₀ (90.00), T₁ (25.33), T₂ (17.83) and T₃(14.67).

DISCUSSION

According to Table 1, the radial growth of Fusarium oxysporum f. sp. psidii was inhibited at high concentrations of carbendazim 50% WP and at low concentration of carbendazim 50% WP the radial growth of Fusarium oxysporum f. sp. psidii was significantly less. This might be due to carbendazim 50% WP is a systemic fungicide which is effective against Fusarium oxysporum f. sp. psidii. Suhag, 1976 reported control of wilt by severe pruning and then drenching with 0.2% either benlate or bavistin 4 times in a year and spraying twice with metasystox and zinc sulphate. Misra and Pandey (1999) reported that though different fungicides viz., bavistin, topsin M, indofil M-45, thiram, blitox check the various wilt pathogens increases its aggressiveness with profuse spore mass production in the soil, once the effect of these fungicides diminishes in soil.

According to Table 2 observations, Trichoderma sp. produced maximum inhibition zone followed by Aspergillus niger and might be due to Penicillium sp. against Fusarium oxysporum f. sp. psidii. This Trichoderma sp. produced toxic diffusates, gliotoxin and because of its fast growing nature it inhibits the growth of the pathogen. Singh et al. (2003), Misra (2006) and Gupta et al. (2009) reported that bioagents like Aspergillus niger, Trichoderma sp. and Penicillium citrinum and some bio-dynamic antagonists have shown their effectiveness towards the control of wilt pathogens of guava. Misra et al. (2004) was also tested these fungi for the control of wilt pathogen in laboratory conditions, these were found quite effective. When relative growth of the three bioagents was studied by Misra and Prasad (2003) then they were found that Aspergillus niger was fastest growing and most effective.

The radial growth of Fusarium oxysporum f. sp. psidii was significantly less in neem leaf extract treatment followed by Lantana leaf extract treatments. Datura leaf extract was not much effective against Fusarium oxysporum f. sp. psidii. Singh et al. (1993), Shivpuri et al. (1996), Bansal and Gupta (2000) and Dwivedi and Shukla (2000) studied and reported that aqueous leaf extracts of Azadirachta indica, Lantana camera, Ocimum sanctum, Datura fastosa, Ficus religiosa, Vitea megendo, Atropa belladonna, Calotropis procera, Eucalyptus amygdalina, Alianthus excels, Vinca rosea, etc. were tested against Fusarium oxysporum. Among these leaf extracts of neem was highly toxic to Fusarium oxysporum showing complete inhibition of mycelial growth and spore germination at 100%. Lantana camera also inhibited mycelial growth and spore germination at different concentrations although they did not exhibit complete inhibition even at 100% concentration.