An attempt of biocontrol the tomato-wilt disease caused by Verticillium dahliae using Burkholderia gladioli pv . agaricicola and its bioactive secondary metabolites

There is a great interest in discovering new microbial natural biocides such as microbial secondary metabolites to reduce the environmental pollution due to the excessive use of synthetic pesticides. Verticillium wilt, caused by the soil-borne Verticillium dahliae, is a widespread disease in tomato growing in many parts of the world. Burkholderia gladioli pv. agaricicola produces some antimicrobial substances and extracellular hydrolytic enzymes which exhibited promising antimicrobial activity towards several phytopathogens. The aims of the current research are to assess in vitro fungicidal effect of 4 strains of B. gladioli pv. agaricicola (ICMP11096, 11097, 12220 and 12322) against V. dahliae using culture or cell-free culture filtrate. In situ assay was performed to evaluate the biocontrol effect of the most efficient bacterial strain on wilt disease caused by V. dahliae in tomato plants. Results demonstrated that the studied bacterial strain ICMP12322 exerted the highest in vitro antifungal activity against V. dahliae which correlated with its ability to produce extracellular hydrolytic enzymes. Furthermore, in situ results showed that the selected bacterial strain significantly minimized the disease incidence.


Introduction
Verticillium dahliae Kleb.(V.dah), the causal agent of verticillium wilt disease, is a soilborne fungus widely spread among a variety of economically important crops. 1 The most common hosts of V.dah are trees, herbaceous ornamentals and vegetables such as tomato, eggplant and lettuce. 2,3Controlling V.dah is difficult because of the persistence of its microsclerotia in the soil. 4V.dah is considered a serious pathogen for the Solanaceae family especially for tomato. 5tually, the isolates of V.dah have been differentiated into two races by virulence assay 6 which are able to infect different hosts such as tomato and lettuce. 1,7,8The above two races of V.dah were differentiated also by molecular methods (PCR assay) using a specific primer VdIGSF1 and VdIGSR1. 9ifferent microorganisms exhibited a biocontrol effect against several phytopathogens depending on their antagonistic action through many mechanisms, such as, synthesis of antibiotics, cell-wall degradation enzymes, and production of siderophores. 10,11he immune system of the plants can be enhanced locally or systemically by biological agents or abiotic inducers, with or without pathogen infection. 12][15][16][17] The bioactive metabolites produced by B. gladioli Zopf were able to significantly inhibit the conidial germination of Penicillium digitatum, P. expansum and Botrytis cinerea. 18,19Further studies reported that the biocontrol mechanism of B. gladioli in vivo could be explicated by the synergic combination between competition for nutrients and/or space and production of antimicrobial metabolites. 16. gladioli pv.agaricicola Yabuuchi (Bga) is an important pathogen of mushroom because it causes soft rot disease of the fleshy tissues of Agaricus bisporus and Pleurotus eryngii. 20,21Several researchers reported the fungicidal effect of Bga against some phytopathogenic fungi such as B. cinerea, Aspergillus flavus, A. niger, P. digitatum, P. expansum, Sclerotinia sclerotiorum and Phytophthora cactorum. 17,20 recent study conducted by Elshafie et al. 11 demonstrated that 4 strains of Bga possess antimicrobial properties and are able to control some phyto and human pathogens such as Bacillus megaterium and Escherichia coli.In addition, Bga produces three important hydrolytic enzymes (chitinase, protease and glucanase) which were suggested to play an essential role in its biological effect. 22he objective of this study was: i) to evaluate the in vitro antifungal activity of 4 strains of B. gladioli pv.agaricicola ICMP11096, 11097, 12220 and 12322 against V. dahliae, ii) to assess the effectiveness of using the most efficient strain in controlling the incidence and severity of verticillium wilt disease on tomato plant.

Tested bacterial and fungal strains
The studied B. gladioli pv.agaricicola was obtained from International Collection of Microorganisms from Plants (ICMP) (Landcare Research, Auckland, New Zealand).The tested strains, ICMP11096, ICMP11097, ICMP12220 and ICMP12322, were maintained as lyophils at 4°C and subcultures were obtained on King Agar B medium (KB) for 48 hr at 22±2°C. 23he V. dahliae strain (no.465) used in this study, previously identified as Race 2 using molecular methods, 24 was isolated from symptomatic eggplant (Solanum melongena L.) and maintained in mycotheca at the School of Agricultural, Forestry, Food and Environmental Sciences (University of Basilicata, Potenza, Italy) on potato dextrose agar (PDA) at 8°C.

In vitro fungicidal activity
The antifungal activity of the above mentioned 4 strains of B. gladioli pv.agaricicola against V. dahliae was carried out by growing the studied bacterial strains on KB medium and incubated at 25±2°C for 24 h.Then 0.5 cm 2 agar disc was placed onto the surface of PDA plates following the bacterial disc method as reported by Tomas et al. 25 The plates were incubated at 22±2°C for 4-5 days.
The antifungal activity of cell-free filtrate of the above 4 bacterial strains was conducted using the diffusion method. 26rlenmeyer flask containing 150 mL of liquid minimal mineral medium (MM) were inoculated with 1.5 mL bacterial suspension containing 10 8 CFU/mL and incubated in rotary shaking (180 rpm) at 22±2°C for 7 days.The culture was centrifuged at 20,000 g for 15 min and filtered using a Millipore membrane (0.20 μm).Fifty μL of the previous filtrate was deposited on 14 mL solid PDA medium inoculated with 0.5 cm 2 of fungal disc.After 4-5 days of incubation, the diameter of the fungal mycelium was measured in mm.

In situ experiment
On the basis of the results obtained from in vitro study, the biocontrol effect of the most efficient strain, was evaluated in situ experiment against V. dahliae.Seeds of Solanum lycopersicum L. cv.cerasiforme were surface sterilized by ethanol (70%) and then were sowed in cell tray for 15 days.The seedlings were transferred to experimental pots for further treatment as follows.

Bacterial suspension treatments
A bacterial suspension containing 10 6 CFU/mL of Bga ICMP12322 was prepared in sterilized broth of Minimal Mineral media (MM) from an initial vegetative culture on solid KB media and was incubated for 5 days at 22±2°C. 23The broth culture was then poured into the pots surface (100 mL/ pot) of about 15 days after sowing (DAS).

Artificial fungal infection
A small portion of fungal mycelium with microsclerotia of V. dahliae was inoculated in sterilized potato dextrose broth (PDB) and incubated for 7 days at 22±2ºC.Later on, 50 ml of the prepared broth was inoculated in the soil near roots zone 22 days (DAS).

Assessment of disease incidence
The plants were monitored daily to observe the eventual appearance of disease symptoms.The disease incidence was assessed using the following scale: 0= no symptoms observed; 1 = 1 to 20% of leaf superficial chlorosis; 2 = 21 to 50% of leaf superficial chlorosis; 3 = 51 to 80% of leaf superficial chlorosis; 4 = >80% of leaf superficial chlorosis.
The infection percentage (IP%) was measured by using equation 1.The disease index (DI%) and the control effect (CE%) were calculated with equations 2 and 3, respectively using the formulas described by Lee et al. 27

In vitro antifungal activity
Results demonstrated that all studied bacterial strains were able to significantly inhibit the mycelium growth of V.dah in plates (Table 1).Furthermore, the bioactive secondary metabolites produced in cell-free culture filtrate were able to significantly reduce the fungal mycelium growth of   V.dah after 5 days of incubation (Table 1).The antifungal activity derived from both strains ICMP12322 and ICMP11096 were significantly the highest.For the current study, strain ICMP12322 was selected for further in situ experiment.

Reduction of disease symptoms effect
The artificial infection of V.dah led to the development of leaf yellowing symptoms within 8-12 days after infection (DAI).Full leaf wilting was monitored after 25 DAI, where the infection percentage was evaluated by 50% for treated plants (positive control) as illustrated in Figure 1.The plants inoculated only with V.dah showed the highest significant disease index estimated by 75.83% compared to the control plants and to those treated only with Bga (1.56% and 1.98%, respectively).
Whereas, the bacterized plants which were artificially infected by V.dah showed a control effect insignificantly close to the negative control plants as illustrated in Table 2.The application of ICMP12322 Bga strain seemed to trigger an acquired plant systemic resistance (SAR).
Biological control using antagonistic bacteria and fungi would have a great environmental interest and can effectively replace the traditional synthetic agrochemicals to control many phytopathogens.Recently, several Pseudomonas, Burkholderia and Bacillus species have been tested to control some crop diseases and showed promising biological effects compared to agrochemicals. 10,16,28egarding the present study, the appearance of wilted leaves on the V.dah treated tomato plants gave evidence for pathogenic effect of V.dah on epigeous and hypogenous portions of the Solanaceae family.Results proved the biocontrolling effect of the studied bacterial strain lead to the high reduction of wilt symptoms on tomato seedlings.The application of bacterial Bga strain could induce a major resistance effect against V.dah.
Similar researchers reported that the use of some PGPB strains such as B. pumilus, B. subtilis and Curtobacterium flaccumfaciens induced significant disease protection against Colletotrichum orbiculare, P. syringae pv.lachrymans and Erwinia tracheiphila. 29Hossain et al. 12 reported that a novel endophytic bacterium B. oryzicola YC7007 was able to significantly inhibit the bakanae disease of rice caused by Fusarium fujikuroi by inducing systemic resistance and antibiotic production.Similar, Chung et al. 28 also reported that an endophytic strain of B. oryzicola has potentially suppressed the bacterial blight and grain rot of rice caused by Xanthomonas oryzae pv.oryzae and B. glumae, respectively, via resistance induction.
On the other hand, several researchers studied the synergic effectiveness of using more than one biocontrol agent, such as mixtures of fluorescent pseudomonads showed a potential suppressive effect against the incidence of Gaeumannomyces graminis var.tritici. 30Although the potential effect of the current studied bacterial strain to control V.dah in tomato plant is adequate in conventional agriculture, however, the combinations between different biological agents and chemical controls may achieve the effective disease suppression on the short term.

Conclusions
The disease index calculated for bacterized tomato plants challenged with V.dah was significantly reduced compared to the positive control indicating that tomato plants treated with Bga ICMP12322 exhibited a significant disease resistance.In conclusion, the results presented here indicate that the application of Bga strain 12322 can enhance disease protection and improve the consistency of biological control against wilt disease caused by V.dah.Further studies are still needed to assess the effectiveness of the studied strains as well as other Bga strains to be used in open field experiments and to study the biocontrolling effect of the combination between more than one biological and/or agrochemical agent.

Figure 1 .
Figure 1.Symptomatic leaves percentage of tomato due to V.dah infection.Bars with different letters indicate mean values significantly different at P<0.05 according to Tukey B test.Data are expressed as mean of 3 replicates ± SDs.IP = [(Symptomatic leaves / Total number of leaves) × 100].

Table 1 . In vitro antifungal activity of 4 Bga strains (cultures and cell-free culture fil- trates) against V.dah in PDA plates. Bacterial strains Mean diameter of V.dah mycelium growth (mm ± SD) Cell cultures Cell-free filtrates
Bga = Burkholderia gladioli pv.agaricicola; V.dah = Verticillium dahliae.Values followed by different letters in each vertical column are significantly different at P<0.05 according to Tukey B test.Data are expressed as mean of 3 replicates ± SDs.

Table 2 . Disease index and control effect of plants subjected to different tested treat- ments.
Bga = Burkholderia gladioli pv.agaricicola ICMP12322; V.dah = Verticillium dahliae; DI% = disease index; CE% = control effect.Values followed by different letters are significantly different at P<0.05 according to Tukey B test.Data are expressed as mean of 3 replicates ± SDs.