Abiotic Biological Control Agents for Crop Disease Management

Abstract

Biological management of crop diseases can be achieved to the desired level by judiciously integrating the biotic and abiotic agents with different mechanisms of action on the target pathogen(s). A wide range of sources of abiotic agents has provided the materials for enhancing the activities of biotic biocontrol agents, resulting in effective disease suppression. Addition of organic amendments such as composts and green manures is known to increase the population and activities of microbial communities that positively influence plant growth and protection offered to plants against microbial plant pathogens. The biosurfactant function of brassica crop amendments has been demonstrated to reduce the incidence and severity of soilborne diseases. Plant extracts and secondary metabolites of plants, like essential oils are effective in reducing pathogen population and disease incidence of soilborne and foliar diseases. Fumigation with thymol has the potential for use as an alternative to chemicals used for the management of postharvest diseases. Naturally-derived compounds like chitosan have high level of biocontrol potential, primarily by enhancing resistance of plants and harvested produce against microbial pathogens. Plant activators such as salicylic acid and acibenzolar-S-methyl (ASM) have been demonstrated to possess marked potential for protecting plants against a wide range of microbial plant pathogens. Phosphates are inexpensive inorganic chemicals that may increase the level of resistance in treated plants to diseases. Likewise, silicon is another inorganic source of resistance inducers, deserving wider exploitation for disease management. Abiotic biocontrol agents are available in large numbers, awaiting recognition by discerning discoverers for realizing their potential.

Biological management of crop diseases has been demonstrated to be ecofriendly and to have the potential to reduce or replace the use of chemicals against diseases caused by microbial plant pathogens. A wide range of materials of plant and animal origin have been evaluated for their efficacy in suppressing the development of microbial plant pathogens and the diseases incited by them. Some synthetic compounds also have been tested for their potential. Organic or inorganic compounds from naturally-derived sources and they have been demonstrated to have potential for effective biocontrol of crop diseases, when applied in the soil and/ or on the seeds and plants.

Appendices

Appendix 8.1: Induction of Resistance to Apple Fire Blight Disease by Acibenzolar-S-Methyl (ASM) and DL-3-Aminobutyric Acid (BABA) (Hassan and Buchenauer 2008)

8.1.1 A. Application of Inducers of Disease Resistance

• i. Prepare solutions of BABA at a concentration of 1.0 mg/ml and ASM at a concentration of 0.1 mg/ml; spray BABA at 4 days before inoculation (dbi) or ASM at 2 dbi separately or spray the inducers simultaneously and spray the control plants with sterile distilled water.

• ii. Inoculate the apple seedlings treated with inducers/control with a suspension of the pathogen (1 × 10⁸ CFU/ml).

• iii. Spray the apple seedlings with BABA and ASM at different days, as determined to select the time interval for optimal expression of acquired resistance.

8.1.2 B. Evaluation of Disease Severity

• i. Inoculate the plants using sterilized scissors dipped in bacterial suspension for 20 s and cut the leaf tips (about 1.5 cm from the tip of two young leaves beneath the two apical ones) of apple seedlings; place the inoculated seedlings in dew chamber at 100 % RH and 24 ± 2 °C for 24 h and transfer the plants to the greenhouse.

• ii. Record the disease indices at 10 days after inoculation expressed as browning discoloration index (BDI) and stem bending index (SBI), as percentages as detailed below for treatments and controls.

Brown discoloration index (BDI) using 0–6 scale: 0 = no leaf symptom; 1 = 25 % or less; 3 = 50–75 %; 4 = >75 %; brown discoloration from the edge of the cutting to the midrib; 5 = >75 % brown discoloration from the edge of cutting to the midrib and/ or the petiole turns brown; 6 = petiole turns brown or black releasing bacterial ooze:

$$ \begin{array}{l}\text{BDI}(\%)={\displaystyle \sum }(\text{number}\ \text{of}\ \text{leaves}\ \times \text{symptom} \text{category}\ )\times 100/\text{number}\ \text{of}\ \text{leaves}\\ \text{evaluated}(2)\times \text{maximum}\ \text{score}\ 6)\end{array}$$

Stem bending index (SBI) using 0–5 scale: 0 = seedlings and leaves fully turgid; 1 = leaf lamina flaccid, stem turgid; 2 = stem bent 0–30° from vertical position; 3 = stem bent 30–60° from vertical position; 4 = stem bent 60–90° from vertical position and 5 = stem bent >90° from vertical position

$$ \text{SBI}(\%)=\text{Category}\ \text{of}\ \text{symptom}\times 100/\text{maximum}\text{socre}(5)$$

Appendix 8.2: Induction of Systemic Acquired Resistance (SAR) by Acibenzolar-S-Methyl (ASM) in Tobacco Against Tomato spotted wilt virus (Mandal et al. 2008)

8.2.1 A. Treatment of Tobacco K326 Plants with ASM

• i. Dissolve ASM (as 50 % active ingredient (a.i) in wettable powder formulation) in water; spray the solution of ASM with a suitable sprayer on K326 plants at 40–45 days after sowing (DAS) with different concentrations of ASM (0–4.0 g a.i./7,000 plants); wash the plants by spraying with 80 ml of water per flat, containing nine plants to move ASM in the root zone.

• ii. To determine the time required for SAR activation, treat the tobacco plants with two concentrations of ASM at 2 and 4 g a.i./7,000 plants; to determine the effect of age of tobacco plants on SAR activation, treat the seedlings (47 DAS) and older plants (77 DAS) with 2 g a.i./7,000 plants.

• iii. Spray the plants with distilled water for non-treated control plants.

8.2.2 B. Challenge Inoculation with TSWV

• i. Challenge the ASM-treated plants with mechanical inoculation with TSWV inoculum; prepare the inoculum by grinding systemically infected leaves in 0.1 M phosphate buffer, pH 7.0, containing 0.2 % Na₂SO₄ and 0.01 M mercaptoethanol at the rate of 1:10 tissue and buffer ratio (w/v); remove the debris by squeezing the extract through a layer of nonabsorbent cotton; add 2 % carborundum 320 grit and 1 % celite 545 to the inoculum and maintain the inoculum on ice.

• ii. Apply inoculum to two youngest fully expanded tobacco leaves and gently rub with cotton swab dipped in the inoculum; gently wash the inoculated leaves with water and maintain the plants in the greenhouse at 25–30 °C.

8.2.3 C. Assessment of TSWV Infection in ASM-Treated Plants

• i. Observe the experimental plants for the development of symptoms of infection by TSWV; count the number of local lesions on two inoculated leaves of each plant at 6 days post-inoculation (DPI); confirm the infection of TSWV by performing ELISA tests, using tenfold dilution of the sap to relative levels of TSWV in the inoculated leaves of different treatments.

• ii. Allow the plants to grow for a longer period for the development of systemic symptoms induced by TSWV.

• iii. Determine the presence and concentration of TSWV in the roots and newly emerged leaves by ELISA tests.

• iv. Measure the plant height and root length to determine the effect of ASM on plant growth and also record the phytotoxicity symptoms, if any.

Appendix 8.3: Induction of Systemic Acquired Resistance in Pea Against Rust Disease by Abiotic Chemical Inducers (Barilli et al. 2010)

8.3.1 A. Application of Inducers of Disease Resistance

• i. Prepare predetermined concentrations of the abiotic chemical inducers [SA (5, 7, 8.5 and 10 mM), BTH (1, 5 and 10 mM) and BABA (5, 10, 20 and 50 mM)] in sterile water; add Tween 20 (0.03 % v/v); apply three droplets (15 μl each) of each inducer on the first leaflets and treat control plant leaves similarly with sterile water + Tween 20.

• ii. Inoculate plants at 5 days after treatment with inducers by dusting freshly collected urediospores of Uromyces pisi (2 mg of spores/plant) mixed with talc (1:10) using a spore settling tower; incubate the plants for 24 h at 20 ± 2 °C in complete darkness and 100 % RH and place the plants in growth chamber for development of disease symptoms.

8.3.2 B. Examination Under Light Microscope

• i. At 24 h after inoculation, cut one leaf per plant at first node; lay them individually adaxial surface up, on filter paper dipped on fixative acetic acid/ethanol (1:3, v/v) mixture to remove chlorophyll from the chloroplast membranes; bleach the leaf segments using several changes of the fixative and transfer to filter paper moistened with tap water for at least 2 h, to soften the tissues.

• ii. Transfer the leaf segments to lactoglycerol (1:1:1, lactic acid/glycerol/water, v/v/v) for at least for 2 h; add a drop of Trypan blue in lactoglycerol (0.1 %, w/v) on a cover glass; place the leaf segment carefully and mount in lactoglycerol on microscope slide.

• iii. Examine approximately 150 urediospores per leaf sample under the microscope (×40 magnification) and count the number of germinated spores (with germtubes at least as long as the diameter of the spore).

8.3.3 C. Assessment of Disease Severity

• i. Record the infection frequency (IF) on first, second and third pair of leaves separately by counting the number of pustules/cm² in a marked area in each leaflet, using a hand lens (×7); convert the number of pustules in the control as equivalent to 100 % and calculate the relative values for the treatments, in relation to that of the control.