Detection and Identification of Fungal Biological Control Agents

Abstract

Microbial plant pathogens constitute an important group of microorganisms capable of causing countless economically important diseases of various crops resulting in heavy quantitative and qualitative losses. Application of fungi for the suppression of the development of diseases incited by microbial pathogens is an ecofriendly disease management strategy receiving acceptance of all concerned with crop production and marketing. Fungi belonging to different taxonomic groups have been demonstrated to have antagonistic potential against one or more plant pathogens to varying degrees. Mycorrhizal fungi have been evaluated for the biocontrol potential and plant growth-promoting effects on the plans treated with them. In order to select the most effective species/strains/isolates, it is essential to assay the biocontrol potential of all isolates by different techniques in in vitro and under greenhouse and field conditions. Simultaneously the isolates/strains with high level of antagonistic activity have to be precisely identified and differentiated from less effective ones. Morphological, biological, biochemical, immunological and genomic characteristics are studied in detail for precise identification and differentiation of fungal biocontrol agents. Polymerase chain reaction-based techniques have been shown to provide rapidly results that are precise, reproducible and consistent. However, the nucleic acid-based techniques do not discriminate live and dead cells of the fungi making it difficult to determine the population of the biocontrol agents that are biologically active. It will be desirable to have results of biological assays to corroborate the results obtained with nucleic acid-based techniques to arrive at meaningful conclusions.

Appendices

Appendix 2.1: General and Selective Media for Isolation of Fungal Biocontrol Agents

• A. General media

• 1.

  Czapek Dox agar	Solution A
  Sodium nitrate	40 g
  Potassium chloride	10 g
  Magnesium sulphate (hydrous)	10 g
  Ferrous sulfate (hydrous)	0.2 g
  Distilled water	1 l
  Solution B
  Dipotassium hydrogen phosphate	20 g
  Distilled water	1 l
  (store the solutions A and B separately in a refrigerator)
  Prepare the mixture of A and B
  Stock solution A	50 ml
  Stock solution B	50 ml
  Distilled water	900 ml
  Sucrose (analar)	30 g
  Oxiod agar No.3	20 g
  Just before autoclaving and for 1 l
  Zinc sulfate (1.0 g/100 ml water)	1.0 ml
  Cupric sulfate (0.5 g/100 ml water)	1.0 ml

• 2.

  Malt extract agar
  White bread malt extract	20 g
  Oxoid agar No.3	20 g
  Tap water	1 l

• 3.

  Oat agar
  Oat meal ground	30 g
  Oxoid agar No.3	20 g
  Tap water	1 l

• 4.

  Potato carrot agar
  Grated potato	20 g
  Grated carrot	20 g
  Oxoid agar No.3	20 g
  Tap water	1 l

• 5.

  Potato dextrose agar
  Potato	200 g
  Oxoid agar No.3	20 g
  Dextrose	15 g
  Tap water	1 l

• i. Potato dextrose agar (PDA) – for Mucodor albus and M. roseus (Stinson et al. 2003); PDA broth – Trichoderma atroviride (McLean et al. 2005).

• ii. Potato dextrose agar (PDA) (Anand and Reddy 2009) amended with colloidal chitin 0.2 % (or) amended with Sclerotium rolfsii cell wall chitin 0.2 %

• iii. PDA amended with chitosan (de Capedeville et al. 2002) chitosan concentrations 0.25–2.0 % (w/v) Adjust with acetic acid to pH 6.0

• iv. For fungi (Jensen et al. 2004) PDA amended with Triton X-100 2.2 g/l; chloramphenicol 0.5 g/l Potato carrot agar amended with chloramphenicol 0.5 g/l Low-nutrient agar (ASDA Micro)

• v. For yeasts Acidified yeast medium (YM) pH 3.7 Osomophilic YM amended with glucose 40 % pH 4.5

• 6.

  V8 agar medium (Chen and Fernando 2006)
  V8 juice	200 ml
  CaCO3	0.75 g
  Agar	15.0 g
  Distilled water	800 ml

• 7.

  Komada’s selective medium for Fusarium spp. (Arie et al. 1995)
  K2HPO4	1 g
  KCl	0.5 g
  MgSO4. 7H2O	0.5 g
  Fe-EDTA	10 mg
  L-asparagine monohydrate	10 mg
  D (+) galactose	2.0 mg
  Pentachloronitrobenzene (PCNB)	0.75 g (a.i)
  Sodium chlorate	0.5 g
  Sodium tetraborate Decahydrate	1 g
  Chloramphenicol	0.25 g
  Agar15 g
  Distilled water	1 l

• 8.

  Nutrient yeast dextrose agar (NYDA) for yeast BCAs (Droby et al. 2002)
  Nutrient broth	8.0 g
  Yeast extract	5.0 g
  D-glucose	10.0 g
  Agar	20.0 g
  Distilled water	1 l

• 9.

  Solid medium for selecting BCAs (Khan et al. 2001)
  Corn steep liquor	10.0 g
  Yeast extract	1.0 g
  KH2 PO4	2.0 g
  K2H PO4	2.0 g
  NaCl	0.1 g
  Glucose	15.0 g
  Malt yeast extract	3.0 g
  Malt extract	3.0 g
  Peptone (type IV)	5.0 g
  Tryptic soy broth
  agar (1/5 concentration)	pH 6.8
  Difeo Lab.Detroit)
  Distilled water	1 l

• 10.

  Fungal endophytes: Oat meal agar (OMA) for Phialocephala fortinii (Narisawa et al. 2004)
  Oat meal	10.0 g
  Bacto agar	18.0 g
  Mg SO4. 7H2O	1.0 g
  KH2PO4	1.5 g
  Na NO3	1.0 g
  Distilled water	1 l

• 11.

  Nutrient yeast dextrose agar (NYDA) for Candida sake CPA-1 (Abadias et al. 2003)
  Nutrient broth	8.0 g
  Yeast extract	5.0 g
  Dextrose	10.0 g
  Agar	15.0 g

• 12.

  Rosebengal – Allisan – streptomycin – Previcur (RASP) selective medium (Metcalf et al. 2004)
  (NH4)2SO4	2 g
  KH2PO4	4 g
  Na2HPO4	6 g
  FeSO4.7H2O	0.2 g
  CaCl2	1.0 mg
  H3BO3	10 μg
  MnSO4	10 μg
  ZnSO4	70 μg
  Agar	20 g
  Cellulose powder	5 g
  Distilled water	1,000 ml
  Adjust to pH 4.0 before autoclaving

• 13.

  Modified Nash and Snyder medium (MNSM) for Penicillium oxalicum (Larena and Melgarejo 2007)
  Peptone (Difco)	1.5 %
  KH2PO4	2.0 %
  MgSO4. 7 H2O	0.05 %
  Agar	2.0 %
  After autoclaving add streptomycin	300 ppm
  Pentachloro nitrobenzene	1 : 1,000
  Molal Na Cl	1

• 14. Trichoderma selective TSB medium (Xia et al. 2011) Potato infusion, 200 g; glucose – 20 g; Chloramphenicol – 0.25 g; Rose Bengal – 0.15 g; Pentachloronitrobenzene – 0.15 g; Streptomycin 0.05 g; Baytan – 0.05 g; benomyl 0.0005 g; agar 20 g; distilled water – 1,000 ml

Appendix 2.2: Generation of Antibodies Against Fungi (Banks et al. 1992)

• A. Preparation of antigen

• (i) Prepare spore suspensions using 0.01 % Tween 80; wash thrice by centrifugation; inoculate 1 ml of spore suspension (10⁶ spores/ml) into 100 ml of liquid medium supplemented with NaCl (100 g/l) and incubate at 25 °C for 7 days in the dark by placing the flask with contents on a rotary shaker

• (ii) Transfer the mycelium by filtering into a sintered glass filter; wash with sterile water and then with sterile phosphate-buffered saline (PBS) containing 2.9 g Na₂HPO_4.12H₂O, 0.2 g KH₂PO₄, 8.0 g NaCl and 0.2 g KCl and 1,000 ml distilled water; freeze overnight at – 20 °C; thaw and transfer to centrifuge tubes and dry in vacuum dryer

• (iii) Collect the mycelium and add 50 ml of liquid nitrogen; mince the mycelium in a blender for 1 min and grind in a mortar with a pestle to a fine powder

• (iv) Suspend the mycelial powder in PBS (200 mg in 100 ml); centrifuge at 4,500 rpm (3,000 g) for 10 min at 4 °C and divide the supernatant containing soluble nitrogen into 0.5 ml aliquots and store at – 20 °C

• (v) Estimate the total protein content of the antigen preparation

• B. Production of polyclonal antiserum

• (i) Mix soluble antigen preparation with equal volumes of Freund’s complete adjuvant (Difco) to produce a final protein concentration of the mixture 1 mg/ml

• (ii) Inject rabbits intramuscularly with 1 ml of the mixture at predetermined intervals

• (iii) Bleed the animal at 4 weeks after the first injection and subsequently at 14, 16 and 18 weeks

• (iv) Separate the serum after completion of clotting of blood cells followed by centrifugation

• C. Production of monoclonal antiserum

• (i) Mix soluble antigen preparation with an equal amount of Freund’s complete adjuvant to yield a final protein concentration of 1 mg/ml

• (ii) Inject a BALB/c mouse, after anaesthetization with 0.1 ml of the immunogen intraperitoneally and subsequently at 2, 4, 6 and 8 weeks after the first injection with PBS and remove the spleen after sacrificing the animal by cervical dislocation

• (iii) Carry out fusion of splenocytes with selected myeloma cell line (PS-NS-1-Ag4) at a ratio of 1 × 10⁸ : 5 × 10⁷ by gentle addition of 2 ml of 30 % polyethylene glycol (PEG) (w/v) over 60 s

• (iv) Add 10 ml of warm serum-free RPMI 1640 medium (Gibco) over next 60 s with gentle stirring; add another 20 ml of RPMI and centrifuge for 3 min at 400 g at room temperature

• (v) Suspend the pellet of cells in 50 ml of growth medium (RPMI 1640) with 20 % Myelone fetal calf serum (FCS) (v/v); dispense cell suspension into five 96-well microplates at 100 μl/well

• (vi) Add 110 μl of hypoxanthine aminopterin-thymidine (HAT) medium diluted to 1:50 in growth medium to each well in the fusion plates

• (vii) Add growth medium + HAT on 2, 4, 7 and 10 days by removing 100 μl of fresh medium

• (viii) Screen the hybridoma cells for efficiency of antibody production by indirect ELISA procedure

• (ix) Clone healthy growing hybridoma twice by limiting dilution in a non-selective medium; preserve by freezing slowly in 7.5 % dimethyl sulfoxide (DMSO) and store in liquid nitrogen

Appendix 2.3: Detection of Fungi by Enzyme-Linked Immunosorbent Assay (ELISA) Test (Bossi and Dewey 1992)

• A. Preparation of antigen

• (i) Prepare surface washings of the fungus grown on PDA for 17–20 days at 21 °C, using 5 ml/petridish of phosphate-buffered saline (PBS) containing 8.0 g NaCl, 0.2 g KCl, 1.15 g Na₂HPO_4. 0.25 g KH₂PO₄, and water 1,000 ml at pH 7.2 and remove the wash suspension by suction

• (ii) Centrifuge the wash fluid for 3 min at 13,000 g to remove the fungal debris and dilute the supernatant with PBS to have 10-fold dilutions

• (iii) Remove the high MW carbohydrates and glycoproteins by passing the cell-free wash fluid through a Centricon 30-kDa filter (Amicon No. 4208) to prevent induction of nonspecific antibodies; freeze-dry the filtrate and redissolve the contents in 1 ml of distilled water and use it as the antigen

• B. Enzyme-linked immunosorbent assay (ELISA)

• (i) Coat the wells (in triplicate) in the 96-well microtiter plates with PBS surface washing fluid (50 μl/well) overnight and wash the wells four times allowing two min for each washing followed by a brief washing with the distilled water

• (ii) Air-dry the plates in a laminar flowhood and seal them in a polythene bag and store at 4 °C

• (iii) Incubate the plates successfully with hybridoma supernatants for 1 h, then with a 1/200 dilution of a commercial goat antimouse polyvalent (IgG + IgM) peroxidase conjugate and finally with PBS with 0.05 % Tween-20 (PBST) for 1 h more

• (iv) Add the substrate solution containing tetramethyl benzidine (100 μg/ml) for 30 min

• (v) Maintain the controls in incubated tissue culture medium containing 5 % fetal bovine serum (FBS) in place of hybridoma supernatant

• (vi) Stop the reaction by adding 3 M H₂SO₄ (50 μl/well); determine the intensity of color developed in each well using ELISA reader at 450 nm

• (vii) Absorbance levels more than three times greater than those of controls indicate positive reaction and presence of antigen protein

Appendix 2.4: Characterization of the Antibody Specific for Fungal Biocontrol Agent by Western Blotting Technique (Thornton et al. 2002)

• A. Polyacrylamide gel electrophoresis (PAGE)

• (i) Use the system with 4–20 % gradient polyacrylamide gels (Bio-Rad) under denaturing conditions; denature the samples by heating at 95 °C for 10 min in the presence of β-mercapto-ethanol prior to gel loading

• (ii) Separate the proteins for 1.5 h at room temperature

• (iii) Use prestained broad-range markers (Bio-Rad) as standards for molecular mass determination

• (iv) Stain the gels for total protein with Coomassive brilliant blue

• B. Western blotting

• (i) Transfer the separated proteins electrophoretically to a PVDF membrane (Immuno-Blot PVDF; Bio-Rad) and wash the membranes thrice with phosphate-buffered saline (PBS)

• (ii) Block the non-specific binding areas in the membranes with PBS containing 1 % bovine serum albumin (BSA) for 16 h at 4 °C

• (iii) Incubate membranes with MAb supernatant diluted (1:2) with PBS containing 0.5 % BSA (PBSA) for 2 h at 23 °C and wash the membranes thrice with PBS

• (iv) Incubate the membranes with goat anti-mouse IgM (μ-chain specific) alkaline phosphatase conjugate (1 : 15,000) diluted in PBSA for 1 h and wash the membranes twice with PBS and once with PBST

• (v) Stop the reaction by immersing the membranes in double distilled water and air dry between sheets of Whatman filter paper

• (vi) Label the proteins immobilized on the membranes using a commercial glycoprotein kit as per the manufacturer’s instructions (Bio-Rad)

Appendix 2.5: Detection of Fungal Biocontrol Agents by Enzyme-Linked Immunosorbent Assay (ELISA) (Thornton et al. 2002)

• (i) Use microtiter plates (96 wells); incubate with selected antibody solution (200 μl) for 1 h and incubate with either a goat-anti-mouse polyvalent (IgG IgA and IgM) peroxidase conjugate (1:1,000) or a goat-anti-mouse IgM (μ – chain specific) peroxidase conjugate (1 : 5,000) for further 1 h

• (ii) Transfer tetramethyl benzidine substrate solution to each well and incubate for 30 min

• (iii) Stop the reaction by adding 3 M H₂SO₄

• (iv) Record the absorbance at 450 nm using an ELISA reader

• (v) Wash the wells four times of 5 min each with phosphate buffered saline (PBST) containing 0.02 M PO₄, 0.85 % NaCl and 0.05 % Tween 20 after each incubation mentioned above [steps (i) and (ii)]

Appendix 2.6: Identification of Rhodotorula mucilaginosa by Dot Blot Hybridization Technique (Utkhade and Cao 2005)

• A. PCR amplification

• (i) Extract the DNA from plant and fungal samples using the Fast DNA kit as per the manufacturer’s instructions

• (ii) Perform amplification using specific primers that are specific to septate fungi and yeasts and amplify the sequences of ITS1, 5.8 S ribosomal gene and ITS 2 portions of rDNA; 5′ AACTTGGTCATTTAG AGGAAGTAA (SF-UP 18570) and 5′GTTTCTTTTCCTCGC CTTAT TGATATGG (UN-LO28S-22)

• (iii) Using a thermocycler (Robocyler, Stratagene, CA, USA) carry out PCR reactions in 20 μl volumes with final concentration of reagents as detailed below: 1 × reaction buffer, 100 μM of each of d-NTP, 500 nM of each primer and 1.25 U Taq polymerase and a drop of mineral oil overlaid on the reaction mixture.

• (iv) Carry out 29-cycles at 94 °C for 45 s, at 58 °C for 45 s and at 72 °C for 45 s

• (v) Maintain a positive control with the BCA (R. mucilaginosa) DNA and a negative control with all reagents + distilled water for each run

• (vi) Analyze the PCR products with standard electrophoresis procedure on 1 % minigels, stained with ethidium bromide and visualize under UV light

• B. Hybridization reaction

• (i) Prepare nylon membrane blots (Roche Diagnostics, Canada) using a manifold device (Schleicher and Schuell, Manifold, USA)

• (ii) Prepare aliquots (5 μl) of PCR product denatured in 0.5 M NaOH (500 μl) for 5 min at 37 °C; mix with 12 × SSC (500 μl; saline sodium citrate) and spot 200 μl aliquots onto nylon membrane through the wells of the manifold device

• (iii) Dry the membranes immediately at 120 °C for 30 min to fix the DNA onto the membrane and cool the membrane

• (iv) Prehybridize the membrane for 2.5 h in hybridization buffer containing 6 × SSC 1 % skim milk powder, 0.2 % sodium dodecyl sulfate (SDS) and 0.1 mg/ml of Poly (A) at 42 or 48 °C

• (v) Hybridize the membrane overnight (about 16 h) in hybridization solution containing optimum concentration of probe (0.2 pmol)

• (vi) Wash the membrane twice for 10 min each in 2 × SSC 0.2 % SDS at hybridization temperature and twice for 10 min each in 0.1 × SSC, 0.02 % SDS at the same temperature

• (vii) Incubate the membrane at room temperature for 30 min in a 1:20,000 dilution of anti-digoxigenin AP (Roche Diagnostics, Canada) and wash as per the manufacturer’s instructions; generate the signal using the CDP substrate (Roche Diagnostics) and capture the signal on scientific imaging film (X-OMAT Kodak, Rochester, NY)

Appendix 2.7: Assessment of Biocontrol Activity by In Vitro Tests

• A. Inhibition of growth of fungal pathogens (Weller et al. 1985)

• (i) Mark two lines perpendicular to each other passing through the center of the bottom of a sterile petriplate using a glass marking pencil; pour about 15 ml of potato dextrose agar (PDA) or King’s medium B in each plate; tilt the plate gently so that the medium spreads uniformly in it; allow the medium to set

• (ii) Cultivate the test antagonists in appropriate medium (PDA for fungi and King’s medium B for bacteria or other specific media) for the required period

• (iii) Using a sterile cork borer (6 mm dia), transfer one culture disk of each test antagonist to the plates containing medium already prepared; position the disks at equidistant points near the edges of the lines marked on the plates; incubate for 2 days at room temperature

• (iv) Grow the pathogen in an appropriate medium; using the sterile cork borer (6 mm dia), punch out disks of pathogen culture; place one disk at the center of the plate in which antagonist cultures have already been placed; incubate the plate at room temperature

• (v) Measure the zones of inhibition formed due to the activity of each test antagonist; select the antagonist causing maximum inhibition of the pathogen

• (vi) To determine the spectrum of activity of the antagonist reverse the positions of antagonist and pathogen; place the culture disks of the antagonist at the center of the plate and the disks of pathogens at the periphery of the plate

• B. Leaf disk inoculation technique (Falk et al. 1996)

• a. Preparation of inoculum of pathogen (Plasmopara viticola)

• (i) Inoculate the grapevine leaves from plants (cv. Riesling) grown in the growth chamber by spraying a suspension of sporangia obtained by washing from sporulating lesions; place the inoculated leaves inside the moist chamber in a growth chamber at 21 °C with a 12-h photoperiod

• (ii) Wash the sporulating lesions formed on inoculated leaves with distilled water from a nonchlorinated fluorocarbon aerosol sprayer; adjust the concentration of sporangia to 1 × 10⁵ per ml using a hemocytometer

• (iii) Place the suspension at 22 °C for 20–30 min for the release of zoospores before inoculation onto test leaf disks/leaves

• b. Preparation of the biocontrol agent (BCA) (Fusarium proliferatum isolate G6)

• (i) Multiply the BCA in potato-dextrose-agar (PDA) medium for 2–3 weeks at 20–25 °C; scrape the mycelium from the surface of the medium and homogenize after adding enough sterile water by vortex mixing

• (ii) Filter the suspension of conidia and hyphae through a double layer of cheese cloth; adjust the concentration of microconidia to 1 × 10⁶ per ml using a hemocytometer

• c. Application of BCA and pathogen

• (i) Prepare leaf disks (2 cm dia) from grapevine leaves of plants grown in the growth chamber; arrange 5–6 inverted leaf disks (lower surface facing upward) on moistened filter paper (Whatman No. 3) placed in each sterile petri plate (9 cm diameter)

• (ii) Spray the conidial suspension of the BCA until the leaf disks are wet; maintain suitable control leaf disks sprayed with sterile water alone

• (iii) Allow the droplets to dry; seal the dishes with parafilm and then place them in the growth chamber with a 12-h photoperiod for 24 h at 21 °C

• (iv) After incubation for 24 h, on each leaf disk place five 10 μl drops of the pathogen suspension containing released zoospores; thoroughly blot the moisture on the leaf disk with absorbent tissues at 20 h after inoculation

• (v) Reseal the petri plates and place them in the growth chamber

• (vi) To assess the effect of the BCA, collect the sporangia formed on each leaf disk using 2–3 ml of distilled water, after 7 days of incubation in the growth chamber; calculate the number of sporangia from each leaf disk using a hemocytometer

• (vii) Assess the resporulation of the pathogen visually in each leaf disk and quantify after 24–48 h as done before; repeat the experiment three times

• (viii) To assess the efficacy of the BCA as post-inoculation application, place five 10 μl drops of the suspension of sproangia and zoospores of the pathogen on the undersurface of each leaf disk; seal the plates and incubate

• (ix) Blot out the moisture on leaf disks after 20-h incubation, reseal and place the petri plates in the growth chamber for 5 days

• (x) Place one 10 μl drop of the conidial suspension of the BCA on the sporulating lesion in each leaf disk; blot out the moisture after 20 h; reseal the plates and incubate for 7 days

• (xi) Quantify the sporangia produced in each leaf disk as done in step (vi) above; assess resporulation after 4 days as done in step (vii)

• C. Petal disk assay (Gould et al. 1996)

• a. Preparation of biocontrol agent (Pseudomonas fluorescens)

• (i) Multiply the BCA on 30 ml of Luria-Bertani (Difco) broth for 48 h at room temperature

• (ii) Centrifuge at 3,000 g for 5 min; reject the supernatant; resuspend the pellet in Tween 20 solution (0.03 %) in water

• (iii) Adjust the optical density to 0.3 at 595 nm to have a concentration of 10⁵ CFU of bacterial cells per ml

• b. Preparation of pathogen

• (i) Multiply the pathogen (Botrytis cinerea) in malt extract agar (MEA) for 5 days at 21 °C

• (ii) Transfer conidia, using sterile forceps to 1 ml sterile water kept in a microcentrifuge tube, disperse the conidia by vortexing for 30 s to have a uniform suspension

• (iii) Adjust the concentration of conidia to 10³ per ml using a hemocytometer

• c. Preparation of test flowers and petal disks

• (i) Raise ultrawhite hybrid petunias in a glasshouse from seeds for 3–4 months; do not use any fungicide or pesticide

• (ii) Collect 3rd 4th or 5th fully opened flowers from the floral apex; surface sterilize by immersing them in 0.05 % sodium hypochlorite solution three times; rinse the flowers in 5 changes of sterile water; select flowers undamaged by disease or pest

• (iii) Punch out petal disks (14 mm dia) using a sterile cork borer

• d. Assay of biocontrol activity

• (i) Dip the petal disks in the suspensions of BCAs at the rate of 4 disks per isolate or BCA to be tested; dip the control disks in sterile water with Tween 20 (0.03 %)

• (ii) Place the BCA-treated and control disks individually in the wells of a sterile multiwell plate with 6 lanes of 4 wells each in the following sequence

  Lane 1 – Well 1 – molten MEA medium; wells 2–4-check petal disks

  Lane 2 – Well 1–4 – check petal disks

  Lane 3–6 – One isolate or BCA for each lane

• (iii) Cover the loaded plates with sterile lids; place them in a plastic box lined with wet paper towels

• (iv) Incubate in a lighted, continuous-mist chamber with a relative humidity of about 90 % and a 12-h photoperiod at 15 ± 2 °C for 24 h

• (v) Dispense aliquots of pathogen inoculum at the center of all petal disks in all treatments (lanes 2–6) and to the first well in lane 1 with MEA medium; do not inoculate the petal disks in the three wells in lane 1; incubate in the incubation chamber for 6 days

• (vi) Assess the extent of disease suppression using a 0–4 scale based on the number of petal disks colonized by the pathogen in a given lane allotted for each BCA or isolate

• (vii) Repeat the test, if the pathogen does not sporulate in the well with MEA (lane 1, well 1)

Appendix 2.8: Biopriming of Carrot Seeds with Biocontrol Agent Clonostachys rosea Effective Against Alternaria spp. (Jensen et al. 2004)

• A. Cultivation of antagonists

  Use nutrient-rich media (a) PDA amended with Triton X-100 (2.2 g/l) and chloramphenicol (0.5 g/l) and (b) potato carrot agar amended with chloramphenicol (0.5 g/l) for isolating the fungal biocontrol agents

• B. Seed Coating (Non-primed)

• (i) Scrap the fungal spores on the surface of PDA after adding sterile water (5 ml); filter through nylon mesh (38 μM) and adjust the conidial concentration 1 × 10⁷ CFU/ml

• (ii) Coat the carrot seeds by shaking the seeds (1 g/treatment) with adjusted conidial suspension (4 ml) on a shaker at 130 rpm for 10 min; keep the seeds on filter paper and air-dry in a laminar hood for 1 h

• (iii) For commercial preparation (dry formulations), suspend in sterile water; shake on a vortexer for 1 min; adjust the spore concentration to 1 × 10⁷ CFU/ml and air-dry as indicated above (step ii)

• C. Hydropriming of carrot seeds

• (i) Use aerated water (500 ml) to be imbibed by carrot seeds (50 g) for 16 h; surface-dry the seeds for 1 h; determine water content at regular intervals to bring the moisture contents (MC) to 38 and 40 %

• (ii) Incubate the seeds in plastic containers with two 1-mm holes in the lid at 15 °C for 13 days

• D. Biopriming of carrot seeds

• (i) Prepare the clay inoculum of the BCA (C. rosea); (2.5 g clay inoculum/500 ml water); apply during imbibition of seeds or dust the inoculum onto seed after drying the seeds at 0.01 g inoculum/g seed at 40 % MC

• (ii) Determine the MC during priming at 7 and 14 days by drying 1-g seed samples at 130 °C for 1 h

• (iii) Air dry the bioprimed seeds in a laminar hood overnight at 22 ± 3 °C to an MC of 7.8 ± 0.2 % before use in bioassays and blotter tests

• (iv) Store the hydroprimed and bioprimed seeds in air tight containers at 4 °C

• E. Bioassay of biocontrol potential

• (i) Plant seeds treated with different BCAs and untreated control seeds after washing in coarse sand moistened with tap water (3:1, v/v) kept in plastic boxes, after covering with transparent plastic foil

• (ii) Place the planted boxes in a growth chamber at 20 ± 1 °C with 12-h photoperiod; water the seedlings using an atomizer after 8 and 14 days after planting

• (iii) Record seedling emergence, dead and wilted plants (removed as and when noted) and confirm the post-emergence infection by incubating affected plants in three layers of filter paper under near UV light to induce sporulation of Alternaria spp.

Appendix 2.9: Identification of Ectomycorrhizal Fungi by Polymerase Chain Reaction (PCR) (Kulmann et al. 2003)

• A. DNA extraction

• (i) Use mycelium (0.05–0.2 g) or washed mycorrhizal roots (3.5 mm); transfer the samples to sterile Eppendorf cup (2 ml) filled with silanized sand and a ceramic sphere and extract the DNA with a QiaGen DNEasy DNA Extraction Kit as per the manufacturer’s instructions

• (ii) Purify the DNA using standard electrophoresis in 1 % agarose gel and stain with ethidium bromide along with known standards

• B. PCR analyses

• (i) Use ITS-specific nucleotides ITS1 primer (TCCGTAGGTGAACC TGCGG) and ITS 8 primer (ACAGGCATGCTCCTCGGAA)

• (ii) Carryout the PCR amplification reactions in aliquots of 50 μl containing 20 mM Tris HCl (pH 8.4), 50 m KCl, 1.5 mM MgCl₂, 3 % DMSO, 100 pmol of each primer ITS1, ITS8, 0.4 mM of each dNTP and 2.5 units of Taq polymerase and 0.1~100 ng DNA

• (iii) Use the thermocycler for amplification with an initial denaturation step at 95 °C for 5 min followed by 35 cycles of 95 °C for 5 min followed by 35 cycles of 95 °C for 1 min, 68 °C for 1 min and 72 °C for 1 min and complete the reactions by a final 2 min extension step at 72 °C

• (iv) Resolve the amplicons by electrophoresis in a 1.5 % agarose 7AE gel followed by staining with ethidium bromide