Ampelomyces quisqualis for control of powdery mildew Optimization of nutritional and growth conditions for the production of conidia of Ampelomyces quisqualis Dario Angeli*, Krishana Saharan**, Ilaria Pertot** * Fondazione Edmund Mach, IASMA, S.Michele a/a, Italy ** Department of Environmental Sciences, Univeristy of Basel, Switzerland
E. necator infections on leaves (severity %) 100 E. necator infections on grape in Trentino-AltoAdige region Infection registered at the end of the growing season 90 80 70 60 50 40 30 20 10 0 AGRO BIO-TECH, Gembloux, Belgium 15 December 2014
Powdery mildew control Powdery mildew (PM) is managed under organic viticulture: Agronomic Chemical Alternative and biological Tolerant varieties Site selection Training system Fertilization Sodium and potassium bicarbonate Calcium polysulphide Sulphur Horticultural oils Plant extracts Compost tea Milk and milk derivates BCAs
Potential Biocontrol Agents (BCAs) Mites - Orthotydeus lambi (parasitism) Bacteria Melidossian, 2005 Melidossian, 2005 - Bacillus Subtilis (competition, parasitism, antibiosis) Yeasts - Aureobasidium pullulans (antibiosis) - Verticillum lecanii (parasitism) - Tilletiopsis pallescens (antibiosis + parasitism) Filamentous fungi - Pseudozyma flocculosa (antibiosis) - Trichoderma harzianum (antagonism) - Ampelomyces quisqualis (parasitism)
(B) late infection stage Ampelomyces quisqualis (A) early infection stage Hyperparasite of powdery mildews Colonizes hyphae, conidiophores, chasmothecia Direct penetration of host cells Host cells are killed shortly after pycnidial formation (2-4 days after infection) R.J.Howard
Ampelomyces as a bio-control agent Bio-control agents are rapidly replacing chemical control agents as they are highly specific, eco-friendly and cost effective Ampelomyces quisqualis is well known for mycoparasite & antagonistic properties towards powdery mildew disease on various crops Performance: unsatisfactory efficacy under open-field conditions RH requirements during germination & penetration into the host Slow grow rate and low conidial germination
Objectives of research work Optimize a suitable liquid culture medium for production of Ampelomyces quisqualis strain ITA3 Efficacy and validation Bio-inoculant formulation To enhance the conidial germination using various stimulatory substances Conidial germination Preparation of a conidia formulations Culture medium development Assessment shelf life of conidia formulations under various temperatures
Origin of A. quisqualis strain ITA3 The high aggressive fungus A. quisqualis strain ITA3 (HQ108049) was isolated from Erysiphe necator on grapevine leaves in Italy by Angeli et al. (2009). Grapevine leaves ITA3 (HQ108049)
Flow sheet of formulation development Condia (10 5 )/ml Pre-Culture -I 250 ml Flask; WV=50ml After 8-9 days, 5ml culture broth transfer into PC-II Pre-Culture - II Environment conditions: ph= 6.5 Temp=25 o C Shaking =220rpm Dark, Apply on various crops 500 ml Flask; WV=100ml Conidia mix with carriers Liquid formulation Solid formulation
Liquid-stage production A. quisqualis is known as a slow growing fungus in vitro and high sporulation might be difficult to obtain in liquid culture. The selection of suitable medium and the ways to obtain conidiation is most important, in short time period. (A) Jaggery broth (B) Potato Dextrose Broth
Selection of suitable medium 1. Jaggery broth 2. Malt extract Jaggery = 2 g/l KCl = 1 g/l Yeast extract = 3 g/l Malt yeast extract broth = 75 g/l Yeast extract = 1 g/l (Yu huan Gu, 1998) 3. Potato dextrose broth 4. Czapek broth Potato Dextrose broth = 42 g/l (Sztejnberg et al, 1989) Czapek broth = 35 g/l (Sztejnberg et al, 1989) ph=5.6, with shaking at 150 rpm and dark conditions Condia 10 5 /ml 50 ml
Effect of different liquid culture media Media Maximum DMW (g/l) Conidia yield (number of conidia Time required to achieve maximum ml -1 ) a Growth (d) Conidia yield(d) Jaggery 2.41-13 - Malt extract 2.01-14 - Potato dextrose 1.13 (2.15 ± 0.31) x 10 4 14 10 Czapek 1.07 (1.01 ± 0.25) x 10 2 14 15 Potato extract 1.05 (1.86 ± 0.41) x 10 2 14 13 DMW=dry mycelium weight; working volume 100 ml in 500 ml Erlenmeyer flask; (-)= no conidiation till 16 days. a Values are mean ± SD of three replicates. Initial ph-6.5 and agitation speed 120rpm. Pycnidia clusters in Jaggery Conidia released from pycnidium
Effect of agitation on the best growth medium Effect of agitation speed on mycelium growth and conidia yield of A. quisqualis strain ITA3 under different shaking conditions Agitation speed (rpm) Maximum DMW Y x/s (g g -1 ) µ (per d) Start of conidiation (d) Conidia yield (number of conidia/ml) a Time required to achieve maximum (g l -1 ) Growth Conidia (d) yield (d) 90 1.14 0.31 0.11 - - 14-120 2.57 1.21 0.25 13 (3.15 ± 0.31) x 10 3 14 17 170 3.89 2.21 0.37 13 (4.33 ± 0.56) x 10 4 14 15 220 5.96 3.12 0.45 11 (4.45 ± 0.50) x 10 4 14 15 270 2.89 0.67 0.15 - - 14 - DMW=dry mycelia weight; Yx/s=biomass yield; µ=specific growth rate; jaggery medium working volume 100ml in 500 ml Erlenmeyer flask; (-)= no conidiation till 16 days. Values are mean ±SD of three replicates. Outcome: - High conidia yield between 170 and 220 rpm - High DMW at 220 rpm
Dry Mycelium Weight ( g/l ) ph profile Growth profile of ITA3, agitation at 220rpm 6 8 5 ph 7 4 6 5 3 2 DMW 4 3 1 2 0 0 2 4 6 8 10 12 14 16 Time (days) 1 Outcome: - Growth lag phase was long: 4 days - Dry mycelium weight : 5.96 g/l at 14 days - ph: no more changed with time
Effect of substances on conidia germination Effect of various stimulatory substances on the conidial germination (%) of A. quisqualis ITA3 in liquid medium Treatments Conidial germination (%) Number of conidia a 12h 24h 36h 48h 7 days 9 days JM ( Control) 0.00 0.00 2.35 5,48 (1.1± 0.31)x10 2 (2.8± 0.64)x10 4 JM+ S1 3.23 21.2 67.5 98.1 (5.0± 0.53)x10 4 (6.1± 0.81)x10 5 JM+ S2 1.14 4.11 21.0 22.0 (1.0± 0.11)x10 2 (1.8± 0.21)x10 2 JM+ S3 0.00 1,22 5.46 8.21 (1.9± 0.17)x10 1 (1.1± 0.11)x10 2 JM+ S4 0.00 6.11 12.3 45.1 (1.0± 0.11)x10 3 (1.3± 0.11)x10 4 JM+ S5 4.65 23.1 69.1 77.1 - - *used concentration of various substances 1 g/l, except powdery mildew extract 1 x 10^5 conidia/ ml). a Values are mean ±SD of three replicates. Outcome: - Lag phase reduced from 4 to 2 days - Maximum biomass achieved after 10 days (before 14 days)
Control ITA3 + S5 ITA3 + S2 ITA3 + S1 Assessment: 24 hs, 25 C
Statistical medium optimization 1. Plackett Burman design Total 32 flasks were inoculated with different concentrations for screening of relevant nutrient components Outcome: - Relevant components for DMW production: Jaggery, MgSO4, NH4Cl, Potato extract Software Design-Expert V 5.0.9 2. Response Surface Methodology Total 19 flasks were inoculated with ITA3 to optimize the nutrient components (-1, 0, +1 level) Outcome: - Optimized concentration: Jaggery (22.5 g/l); MgSO4 (0.45 g/l); NH4Cl (0.45 g/l); Potato extract (8 g/l)
Results with optimized medium Outcome: - Biomass increased from 5,96 to 58,2g/L at 8 days - Conidia yield increased from 2,8x10 4 to 1,4x10 6 at 8 days Mycelial growth of ITA3 Conidia released from pycnidia Formation on pycnidia into mycelium
Formulation development Stabilize the organism during production, distribution and storage, so that viability is retained throughout the process Aid in handling and application of the product so that it is easily delivered to the target in the appropriate form & manner Protect the Biocontrol Agent (BCA) from harmful environmental factors at the target site and enhance the efficacy by increasing activity, reproduction, contact and interaction with the target organism Liquid conidia formulations Solid formulation
Conidia survivability in different temperatures (a) Silicagel based formulation (b) Glycerol based formulation Outcome: The viability of conidia decrease with higher temperature For long time formulations storage at 28 C temperature was found a suitable
Conidial germiantion (%) Shelf-life with different carriers Strain ITA3 in different carrier based formulations during 150 days storage at 28 ºC 100 90 80 70 60 50 40 30 20 10 0 a ab b c c a ab b 30 d 60 d 90 d 120 d 150 d c d a b b c Glycerol Skimmed milk Trehalose Sucrose Soyabean Silicagel Experiments were performed in triplicate. Data were assessed every 30 days for 5 months. Initial conidial concentration was 1.5 10 5 conidia ml -1. Values with the same letter (a-e) within a column are not significantly (P > 0.05) different according to the Tukey s HSD test. a c cd d Outcome: Conidia survive well in Silicagel and Glycerol d de bc d de e e a a ab b b
Summary 1. The optimized Jaggery broth significantly increased the maximum biomass (10-fold) and conidia yields (1,4 x10 6 ) in shake flask by reduction of 43% time (from 14 to 8 days) 2. The overall biomass production was 0.344 g/l/h (two times higher than obtained with the original jaggery medium) with kinetic parameters of biomass yield (Y x/s ) 3.29 and specific growth rate (µ) 0.56 d -1 3. Stored conidia formulations of strain ITA3 with different carriers showed significant changes in conidia viability on different temperatures
Significance and Impact of this work The work performed in our laboratory resulted in an optimized and inexpensive culture medium which can be used for large scale fungus mass and conidia production for agricultural and horticultural applications Conidia formulations based on liquid and/or solid carriers can easily be used and they can be a good alternative for long time storage Research is currently underway towards development of conidia formulation strategies for use as effective biocontrol agents against powdery mildews in field conditions
Thank you for attention Autonomous Province of Trento European Union Seventh Framework Programme (FP7/ 2007-2013) - grant agreement no. 265865- PURE