Impact of Some Soil Amendments and Mycorrhiza on Cowpea Damping-off Caused by Rhizoctonia Solani

Seeking for new strategies to control plant diseases and improve the yield led to discover different new ideas, including soil organic compounds amendmens such as biochar and compost. These compounds are well known with their suppressive effect against a wide range of soil borne pathogens (Coventry et. al., 2005 and Noble and Coventry, 2005).

Biochar is an organic product rich in carbon, it produced by a heating process known as pyrolysis (Sohi et. al., 2010; Elad et. al., 2011 and Sparks, 2011). The kind of organic compounds and the temperature used for its production determine its nutrient contents and physicochemical properties (Antal and Gronli, 2003 and Gaskin et. al., 2008). The addition of biochar to the soil improve its characterization resulting in beneficial effects on the plant quality and quantity (Glaser et. al., 2002; Steiner et. al., 2008 and Atkinson et. al., 2010). It is very stable in soil with a half-life up to thousands of years (Zimmerman, 2010). Recently, it has been reported that soil amended with biochar can influence the development of the plant diseases caused by foliar and soil borne pathogens (Graber et. al. 2014).

Another soil amendment with suppressive effects is compost. It inhibits a wide range of plant diseases caused by various soil-borne pathogens. This could be due to an enhanced competition and antagonism by the soil biota associated with increased microbial activity in soil (Pugliese et. al., 2011).

Vascular arbuscular mycorrhizal (VAM) fungi are one of the most important microorganisms. It makes symbiotic relationships with the plants and affect soil borne and foliar pathogens (Whipps, 2004 and Fritz et. al., 2006).

Cowpea (Vigna sinensis Endl.) is the most important vegetable crop. The cowpea seeds contain about 23% protein and 57% carbohydrate (Belane and Dakora, 2009). Cowpea plants are liable to attack by damping-off and root rot diseases caused by Fusarium solani, Rhizoctonia solani, Macrophomina phaseolina, Sclerotium rolfsii and Pythium sp. These diseases cause considerable losses to cowpea plants worldwide (Shihata and Gad El-Hak, 1989; Ushamalini et. al., 1993; Rauf, 2000; Satish et. al., 2000; El-Mohamedy et. al., 2006).

This study evaluate the effect of compost, biochar and mycorrhiza, either alone or in combinations, on control of cowpea damping-off disease caused by Rhizoctonia solani and on some plant growth parameters in the greenhouse and field conditions. Also, the study was expanded to evaluate the effect of these treatments on plant content of nitrogen, phosphorus and potassium (NPK).

1. Isolation of fungi associated with cowpea damping-off disease:

Diseased cowpea seedlings, showing the typical symptoms of damping-off disease were collected from the Experimental Farm of the Fac. Agric., Cairo Univ.

For isolation, infected roots were washed thoroughly with tap water and cut into small fragments (0.5- 1.0 cm), superficially sterilized with 1% sodium hypochlorite for 3 min., Then rinsed several times in sterilized water, blotted to dry between folds of sterilized filter papers. Small pieces were transferred onto PDA medium into Petri plates and incubated at 25±1°C for 7 days. Observations were daily recorded and emerged fungi were picked up and cultured on PDA medium slants and its frequencies were calculated. Fungal growth was microscopically examined and purified using the single spore and/or the hyphal tip techniques (Dhingra and Sinclair, 1985). The purified fungi were identified according to their morphological features, either to the generic or to the species level, according to Booth and Waterston (1964) and Barnett and Hunter (1972). The most frequent fungus was selected after confirming its pathogenic capability.

2. Source of tested biochar, compost and mycorrhiza:

Rice straw biochar and commercial compost were kindly obtained from Soil, Water and Environ. Res. Inst., Agric. Res. Center, Giza, Egypt. The characteristics of compost and rice straw biochar are mentioned in Table (1). For mycorrhiza inoculation, Mycorrhizen was utilized as a commercially available inoculum. This mycorrhiza based product was purchased from Soil, Water and Environ. Res. Inst., Agric. Res.Center, Giza, Egypt.

3. Laboratory Test

3.1. Preparation of Compost Water Extract (CWE)

The CWE was prepared by vigorously shaking of mature compost, at the rate of 1 : 2 (w/v) of compost (500 g) to sterile water (1000 ml), for 20 min. To remove

Table 1. Selected characteristics of compost and rice straw biochar used in the present study

Tested compounds pH Total carbon (%) Total N (%) Total P (%) Total K (%)

Biochar 9.0 36.60 0.52 0.54 0.88

Compost 8.19 25.05 1.31 1.65 -

large particles from compost mixture, aliquot of 250 ml of the mixture were filtrated by passing through sterile 3 layers of cheese cloth and then the filtrate was centrifuged at 500 rpm for 10 min to obtain active supernatant as stock solution. Four different concentrations, i.e. 0, 5, 10 and 15%, were tested against the tested fungus.

3.1.1. Effect of biochar and compost on mycelium growth of R. solani:

The inhibitory effect of the tested compost as water extract (CWE) was examined in vitro against the tested pathogenic fungus using the wells-cut diffusion method according to El-Masry et. al. (2002). The CWE was filtered through 0.22 μm sterilized Millipore membrane filter. Fifteen ml of sterile PDA medium were used for each plate, one bore was made on one side of the plate using a sterile 0.5 cm cork borer, and the bore bottom was sealed with two drops of sterile PDA medium. One ml of each CWE concentration were separately transferred to each well. The sterile water was used as control treatment. Five Petri dishes were used as replicates for each treatment as well as the control treatment. All plates were incubated at 25±1°C for 7 days and the reduction in mycelium growth was recorded.

The toxicity of the tested biochar was studied using an in vitro contact assay to evaluate the reduction in R. solani growth. PDA medium was amended with varying concentrations of biochar, i.e. 0, 0.5, 1 and 3%, w:v before autoclaving and then poured into Petri dishes (9-cm-diam.). Agar plugs (5-mm-diam.), covered with actively growing mycelium, were transferred into the center of Petri dishes amended with one of the four concentrations of biochar and then incubated at 25±1°C for 7 days, then the fungal growth was measured. The fungal growth inhibition (%) was calculated using the following formula: I = C-T/CX100

Where; I= Reduction (%) in fungal growth; C= Fungal growth in the control treatment and T= Fungal growth of treatment

3. Greenhouse experiments:

Effect of compost and biochar at different concentrations on cowpea dampping-off disease under greenhouse conditions:

In order to determine the most effective concentrations of the tested compost and biochar, cowpea seeds (cv. Tiba), obtained from Agric, Res. Center, Giza, Egypt, were surface disinfested in 1% sodium hypochlorite, rinsed in sterile distilled water and then 5 seeds were sown in each plastic pot (30 cm3.) filled with a sterilized mixture of sand and clay (2:1, v/v) containing compost at 0, 5, 10 and 15% w/w or biochar at 0, 0.5, 1 and 3%, w/w. One day later the treated soil was individually infested with the tested fungal inoculum at the rate of 3% w/w, previously grown on sand barley medium (1/1, w/w and 40% water) at 25±1ºC for two weeks. Five randomly replicated pots were used for each treatment.

4. Field experiments:

Effect of compost, biochar and mycorrhiza alone or in combinations on cowpea dampping-off disease under field condition during 2013 and 2014 growing seasons:

The most effective concentrations of the tested compost and biochar were selected to study their effect on the disease suppression when used alone or mixed with mycorrhiza. The following treatments were used in the experimental setup:

1. 1. Compost;
   2. Biochar ;
   3. Mycorrhizen;

• Compost + Mycorrhizen;
• Biochar + Mycorrhizen;
• Compost + Biochar + Mycorrhizen;
• Rhizolex-T;
• Control.

The sterilized cowpea seeds were coated with the mycorrhiza inoculum before sowing in the treatments including mycorrhiza. The experiment was carried out at the experimental unit of Plant Pathology Department, Faculty of Agriculture, Cairo University, Giza, Egypt during two successive seasons of 2013 and 2014. The land was divided into ridges (70 cm width). The seeds were sown at a distance of 15cm in one row on the ridge, one seed in each placement.

Seeds were sown on April 15th, 2013 and 2014 seasons. All agricultural practices were carried out according to the recommendation of Ministry of Agric., Egypt. The experimental treatments were arranged in complete randomized blocks design with three replicates. The plot area was 4 m2 (1 m length and 4 m width).

Percentages of pre- and post- emergence damping-off as well as healthy survived plants were carried out 15, 21 and 45 days after sowing, respectively, using the formula described by Mikhail et. al. (2005) and Abd El-Moneim, et. al. (2012) as follows:

Pre-emergence (%) = Number of non-germinated seeds / Total number of sown seeds × 100

Post-emergence (%) = Number of dead seedlings after emergence / Total number of sown seeds × 100

Survived plants (%) = No. of survived plants / Total No. of sown seeds × 100

Survival efficacy (%) = D1-D2 / D1 × 100

Wherease: D1 = Damping-off (%) in control treatment and D2 = Damping-off (%) in treatment

5. Effect of compost, biochar and mycorrhiza alone or in combinations on some parameters of cowpea plants under field conditions during 2013 and 2014 growing seasons:

The vegetative growth parameters of cowpea plants, i.e. plant height (cm), root length (cm), number of leaves, pods and nodules / plant, fresh weight of leaves and roots (g) and dry weight of leaves and roots (g), were determined 90 days after sowing. Five random samples of cowpea plants representing each treatment were removed carefully from the plots, and then washed under running tap water to remove adhering particles.

6. Effect of compost, biochar and mycorrhiza alone or in combinations on nitrogen, phosphore and potassium content of cowpea plants under field conditions during 2013 and 2014 growing seasons:

Nitrogen and phosphorus contents were assayed according to Jackson (1973), wherease, potassium content was determined using atomic absorption spectrophotometer (Barkin Elmer, 3300) according to (Chapman and Pratt, 1961), the results were calculated as g/100g dry weight.

5. Statistical Analysis

Most of the data were statistically evaluated according to Snedecor and Cochran (1967). Averages were compared at 5% level of probability using the Least Significant Differences (L.S.D.) as mentioned by Fisher (1948). On the other hand, percentages data were transformed to arcsines and then subjected to statistical analysis to determine the least significant differences (L.S.D.) to compare variance between treatments (Gomez and Gomez, 1984).