Abstract: Twelve Psuedomonas fluorescens strains were selected for disease control in greenhouse and field trial. They were screened on the basis of their in vitro antagonistic activity towards Cochliobolus sativus (Cs D-18) and Fusarium graminearum (Fg B-4) the inducers of barley root rots. The selected strains were identified as Biovar1 Psuedomonas fluorescens. P. fluorescens D-3, D-5 and D-8, were the most effective for disease controlling under field conditions, in which significantly (p≤ 0.05) reduce disease severity and increase yield.
INTRODUCTION
Barley root rots caused by several species of fungi such as Gaeumannomyces graminis var. tritici, Cochliobolus sativus, Fusarium graminearum, F. culmorum, Rhizoctonia cerealis, Pythium sp. (Paulitz et al., 2002; Smiley et al., 2005; Wiese, 1987), is an important root rot disease in Mazandaran province Iran.
Among the described causal agents, C. sativus and F. graminearum are the common pathogen of barley root rot, in which yield losses estimated up to 30% (Foroutan et al., 2001).
Recently there have been some reports that, in the areas where barley is the only option for plantation and planted continuously, excessive and repeated use of fungicides is being common. As a result it may spoil the soil, ground water and pollute even the atmosphere. These problems have renewed public interest in exploring alternative or supplementary non-polluting sources of pesticides i.e., the biopesticides.
Since the 1980s rhizobacteria and other microorganisms have been investigated as possible replacements for chemicals used to control a broad range of plant diseases. Isolates from the genus Pseudomonas have been tested due to their widespread distribution in soil, ability to colonize the rhizospheres of host plants and ability to produce a range of compounds antagonistic to a number of serious plant pathogens (Anjaiah et al., 1998; Maurhofer et al., 1991; Rodriguez and Pfender, 1997; Mathivana et al., 2004; Ross et al., 2000). Weller and Cook (1983) reported that 10-25% of the yield increases by the application of root-colonizing bacteria antagonistic to take-all pathogen as a seed treatment at planting time. Raaijmakers and Weller (2001) could suppressed take-all disease by means of seeds coating with the strains Q 8r1-96, Q2-87 and 1M1-96. They also figured out a relationship between take-all suppression and the rhizosphere population density of DAPG-producing Pseudomonas sp.
In Iran, suppression of wheat Take-all by bacterial and fungal isolates was reported by Sedaghatfar et al. (2002) and Foroutan et al. (2001) in greenhouse studies.
Not much study has been conducted on the use of biological agents in control of barley root rot diseases in Iran. Therefore this was the first study in the area, with the aim of finding the species of Pseudomonas existing on barley rhizosphere to replace with chemical control.
MATERIALS AND METHODS
Barley (Hordeum vulgare) seeds of Valfajr facultative cultivar, which is the commercial in the province, Cochliobolus sativus D-18 and Fusarium graminearum B-4 (the most aggressive strains of the pathogens among the other strains), obtained from Agricultural and Natural Resources Research Center of Mazandaran.
For isolation of barley rhizobacteria various samples were collected from different parts of barley fields in Baykola (Neka), Dashtnaz (Sari), Kolet (Neka), Gharakheil (Ghaemshar), Firouzkandeh (Sari), Jouibar, Semeskandeh (Sary) and Zirvan (Behshar) of Mazandaran province in Iran during 2002-2003. The samples were taken from the root zone of one month old barley seedlings. Barley plants were dug out and gently shaken off and then the soil closely adhering the roots were collected. Rhizosphere bacteria were isolated using nutrient agar (NA) and King B Medium (Weller and Cook, 1983). One gram of roots and associated soils was suspended in 5 mL of sterile distilled water and was vigorously shaken for 15-20 min.
| Table 1: | Areas and code of bacterial antagonist isolates |
Serial dilution was done by transferring 1 mL suspension to 9 mL sterile distilled water. A 0.1 mL suspension of 107 to 109 dilutions were spread on solidified either nutrient agar or King B Media. The plates then were kept for incubation at 25 to 28°C for 24 h. The individual bacteria colonies were picked on NA slants and after incubation at 28°C for 24 h, the slants were kept at 4°C for short period maintenance. All bacterial isolates were purified, coded and preserved. The code were made based on locations in which the samples had been collected such as: B (Baykola), D (Dashtnaz), K (Kolet), G (Gharakheil), F (Firouzkandeh), J (Jouibar), S (Semeskandeh) and Z (Zirvan) as showed in Table 1.
The bacteria were screened on the basis of their in vitro antagonistic activity towards Cs D-18 and Fg F-4 (Weller and Cook, 1983). Twelve superior bacteria were selected to disease control in greenhouse and field conditions.
Identification of antagonist bacteria was done on the bases of the ability of isolates to produce fluorescent by plating bacteria on King's medium B, biochemical and physiological tests such as; Gram, hyper sensitive reaction in tobacco, oxidizes, nitrate reduction and fluorescence production (King et al., 1954; Krieg and Holt, 1984; Holt et al., 1994; Schaad et al., 2001).
Disease control
In greenhouse: Valfajr barley seeds were bacterised with 12 superior
bacterial isolates. The seeds were soaked for 4 h in pre-grown selected bacterial
isolates on nutrient broth yeast extract agar (NBYA) separately, at the rate
of 109 colony forming unit cfu mL-1, approximately OD
0.2-0.3 at A620 n (Weller, 1988). Nutrient broth yeast extract agar was prepared
based on 2, 2, 0.5, 2.5 and 1.5 g of nutrient broth, yeast extract, K2HPO4,
K2H2PO4, glucose and agar respectively per
liter distilled water (Baudoin et al., 1988).
The seeds also were coated with Rovral-Ts (2 g kg-1) as selected fungicide. This fungicide was screened following in vitro inhibition assay, among the other candidate fungicides i.e., propiconazole, Benomyl. Fungicide was used in greenhouse and field evaluation studies, to compare the efficacy of bio-control versus chemical control against the disease.
The coated seeds were air dried for 5 h in a laminar flow cabinet, then were planted on pre-inoculated potted soil with Cs D-18 and Fg B-4 separately. There were three replication pods for each treatment, based on a randomized completed design (Balasubramaniyan and Palaniappan, 2004), with 29 treatments. The treatments were include: 1-13. Seed coated with antagonists and fungicide and planted in inoculated soil with Cs D-18, 14. Control 1 (non treated seeds were planted in inoculated plots with Cs D-18), 15-27. Seed coated with antagonists and fungicide and planted in inoculated soil with Fg B-4, 28. Control 2 (non treated seeds were planted in inoculated plots with pathogens Fg B-4), 29. Control 3 (non treated seeds were planted in non inoculated plots with the pathogens).
Effect of antagonists then was done after disease progress on inoculated control plants with the pathogens (control 1 and 2). Disease rating was done on the bases of 0-4 scale, where, 0 = no disease and 4 = death of plants.
In field: The experiment was carried out at the Dashtnaz Agricultural Research Station of Mazandaran. The site was cultivated with an offset disk plough to 15 cm depth and again to a 10 cm depth on Oct.11, 2004. The soil was sterilized with methyl bromide at the rate of 40 g m-2 on Oct.12, 2004 to removal any pre-contamination of the trial soil. Cress plant (Lepidium sativum) seeds were grown as bioassay test to insurance the removal of methyl bromide hazard effects on Nov. 11. 2004.
An experimental design was carried out as randomized complete block (Balasubramaniyan and Palaniappan, 2004), with 29 treatments and in 3 replication, the method was the same as described above. Plots size was 0.9 x 6 m. The trial was received 60 kg h-1 diammonium phosphates, 50 kg h-1 P2O5 and 100 kg h-1 urea. The plots were pre inoculated either with 2 weeks old colonized oat (Avena fatua) seeds with strains Cochliobolus sativus D-18, Fusarium graminearum B-4 separately, or sterile oat (Avena fatua) seeds as control 3.
The trial was sown with bacterised and fungicide coated seeds of Valfajr barley and at the rate of 60 kg ha-1 on Nov. 15. 2004. The seeds were sown in four rows (6 m long and 30 cm of row spacing). The trial was kept weeds free by hand weeding in several times as required.
Population dynamic of antagonists: Samples of barley plants were collected after 1 and 2 months of planting dates. Five to six randomly selected plants were dug out from each replicated at each time. Root samples were prepared for isolation of the bacteria, to determine the population size of antagonists. One gram of roots and associated soils was suspended in 5 mL of sterile distilled water and was vigorously shaken for 15-20 min. Serial dilution and other measurements were done as described above. The numbers of antagonist bacteria colonies were enumerated after 48 h.
Disease evaluation: Disease evaluation, was done after disease progress on inoculated plants with the pathogens (control 1 and 2), on the bases of 0-4 scale, (the same method as described in greenhouse method), in the end of April 2005. Two rows of the each plot were harvested by hand and grain yields were obtained using a small thresher and analyzed on May 24, 2005.
RESULTS
A total of 32 barley rhizobacteria out of 220 isolated from the rhizosphere of different barley fields in Mazandaran province were showed to have inhibition zone toward pathogens on PDA medium. The antagonism of the bacteria was tested following dual culture technique. Twelve out of 32 had maximum inhibition against Cs D-18 and Fg B-4. The selected strains were identified as Biovar 1 Psuedomonas fluorescens. They could significantly (p ≤ 0.05) affect the mycelial growth of the pathogens.
The most effective bacteria include; P. fluorescens B6 P. fluorescens B11, P. fluorescens D3, P. fluorescens D5,
P. fluorescens D8, P. fluorescens F12 P. fluorescens F13, P. fluorescens J 22, P. fluorescens K 4, P. fluorescens N 6, P. fluorescens S 5 and P. fluorescens S 10.
Population dynamic of antagonists and effects of antagonists on infection degree and yield were summarized at Table 2.
Effect of bacterial isolates on disease severity and yield induced by Cs and Fg in greenhouse and field are showed at Table 3.
| Table 2: | Population rates of antagonists on barley roots |
| *non detected | |
| Table 3: | Effect of bacterial isolates on disease severity and yield induced by Cs and Fg in greenhouse and field |
| *Mean of 5 plants /pod **1000 grain weight ***Mean of 5 plants /plot. Mean followed by different letter differ significantly at p ≤0.05 | |
DISCUSSION
Pseudomonas strains have been considered to have an attribute to biological control of some soil borne diseases (Capper and Campbell, 1986; Duffy and Weller, 1994).
In vitro experiments revealed that 12 out of 32 isolates showed antifungal activity and consistently induced reproducible zone of fungal inhibition on PDA medium. These isolates were selected for the antagonistic against the root rots diseases in greenhouse and in field trials. All 12 isolates could reduce of disease severity and increased the grain yield in greenhouse trials. But only 3 isolates (P. fluorescens.D-3, P. fluorescens D-5 and P. fluorescens D-8) were the most effective in controlling the disease under field conditions, but other selected bacteria did not have this ability. Because all factors in which were stabled in green house, could not be controlled in the field trial. Also population size of these three isolates showed their ability in colonizing of root system (Table 2). The 3 isolates as described above are the native and were obtained from the same field in which the study was done. So these strains could successfully adapt to the rhizosphere in that field as reported by Duffy and Weller (1994), Raaijmaker and Weller (2001) and Weller and Cook (1983). Ross et al. (2000) expressed that many biological control agents are found to be active only in certain soil types.
Effects of factors such as soil texture, organic matter, pH, water and oxygen availability and competition for nutrients with indigenous micro flora on dampen the biological activity of introduced inocula, also have been described by Capper and Higgins (1993) and Johnsson et al. (1998).
As conclusion 3 isolates (P. fluorescens. D-3, P. fluorescens D-5 and P. fluorescens D-8) in which were the most effective in controlling of two barley root rot diseases under field conditions, can be used in the integration with other disease management systems to afford greater levels of protection and sustain crop yield.
ACKNOWLEDGMENTS
I would like to place my deepest gratitude towards Agricultural and Natural Resources Research Center of Mazandaran and also Dasht Naz Research Station for their supporting during the study period and for making available all the facilities.