Entomopathogenic fungi to suppress BMSB. Participants: Thomas Pike (Graduate Student), Paula Shrewsbury, Ray St. Leger, Department of Entomology, UMD
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1 Entomopathogenic fungi to suppress BMSB Participants: Thomas Pike (Graduate Student), Paula Shrewsbury, Ray St. Leger, Department of Entomology, UMD
2 Fungi to suppress BMSB BMSB SCRI Grant (3 year, 2014) Subobjective Identification of a single, well characterized strain of fungi expressing the optimum toxin combination for BMSB control Subobjective Develop attract-and-kill and mass trapping strategies for management of BMSB in commercial crops BMSB SCRI Renewal Grant Subobjective Development of BMSB-Specific Fungal Entomopathogens (Ray St. Leger, Department of Entomology, UMD)
3 Fungi that colonize and kill their insect host Effective at controlling insects that won t readily consume topical pathogens Bacillus thuringiensis (Bt) Metarhizium anisopliae, Beauveria bassiana and Isaria fumosorosea among the most common entomopathogenic fungi used Used in insect systems such as grasshoppers, flies, beetles, caterpillars and greenhouse pests
4 Determine the effects of entomopathogenic fungi on brown marmorated stink bug Wild-type fungal strains Additives (diatomaceous earth and horticultural oil) Transgenic fungal strains Explore their potential as biological control agents
5 Strains evaluated: Metarhizium (M): ARSEF 1548, ARSEF 2547, ARSEF 1055, F52 (4 strains) Beauveria (B): GHA, Botanigard (2 strains) Isaria (I): ARSEF 3581 (1 strain) Other (U): ARSEF 10386, Unidentified fungus (isolated from lab colony) (2 strains)
6 Fungi plated on PDA media Spore suspensions of 1x10 7 conidia/ml used with.01% Tween (later DI water) BMSB submerged in suspension, placed in plastic bowls with food and water Stink bugs monitored for mortality Tested on nymphs and adults Not all treatments represented in each bioassay (9 bioassays)
7 Mean % Mortality x % Mortality (Day 3) x % Mortality (Day 7) Bioassay # Treatment Adults Nymphs Adults Nymphs (M) (±3.33) 10 (±4.47) 30 (±4.47)a (M) 3.33 (±3.33) (±6.67) (±9.54)a 1 Tween 3.33 (±3.33) (±3.33) 0b 1 Water 3.33 (±3.33) 0 10 (±4.47) 0b (M) 10 (±10) (±15.20) (±12.01) 100 (±0) 2 GHA Botanigard (B) 3.33 (±3.33) 40 (±7.30) 30 (±11.25) 90 (±6.83) 2 Tween 10 (±6.83) 40 (±13.66) 40 (±15.49) 80 (±7.30) 2 Water 6.67 (±4.21) (±8.43) (±9.88) (±9.88) (M) 3.33 (±3.33) 40 (±7.30) (±8.02) (±4.21) 3 Unidentified Fungus (U) 10 (±6.83) 20 (±7.30) (±9.54) (±4.21) 3 Tween 10 (±4.47) (±6.14) (±8.43) (±6.67) 3 Water (±8.02) (±8.43) (±6.14) 4 F52 (M) 25 (±5.00) 25 (±12.58) 60 (±8.16) 70 (±12.91) 4 GHA USDA (B) 30 (±12.91) 20 (±14.14) 65 (±9.57) 65 (±9.57) (I) 20 (±8.16) 40 (±14.14) 55 (±5.00) 80 (±0) 4 Water 25 (±18.93) 35 (±9.57) 45 (±18.93) 65 (±5.00) (U) (±6.67)b (±8.43)b (I) 30 (±8.56)a (±8.43)a 5 Tween (±3.33)b (±6.67)b 5 Water 3.33 (±3.33)b (±6.67)b
8 Mean % Mortality x % Mortality (Day 3) x % Mortality (Day 7) Bioassay # Treatment Adults Nymphs Adults Nymphs (M) (±3.33) 10 (±4.47) 30 (±4.47)a (M) 3.33 (±3.33) (±6.67) (±9.54)a 1 Tween 3.33 (±3.33) (±3.33) 0b 1 Water 3.33 (±3.33) 0 10 (±4.47) 0b (M) 10 (±10) (±15.20) (±12.01) 100 (±0) 2 GHA Botanigard (B) 3.33 (±3.33) 40 (±7.30) 30 (±11.25) 90 (±6.83) 2 Tween 10 (±6.83) 40 (±13.66) 40 (±15.49) 80 (±7.30) 2 Water 6.67 (±4.21) (±8.43) (±9.88) (±9.88) (M) 3.33 (±3.33) 40 (±7.30) (±8.02) (±4.21) 3 Unidentified Fungus (U) 10 (±6.83) 20 (±7.30) (±9.54) (±4.21) 3 Tween 10 (±4.47) (±6.14) (±8.43) (±6.67) 3 Water (±8.02) (±8.43) (±6.14) 4 F52 (M) 25 (±5.00) 25 (±12.58) 60 (±8.16) 70 (±12.91) 4 GHA USDA (B) 30 (±12.91) 20 (±14.14) 65 (±9.57) 65 (±9.57) (I) 20 (±8.16) 40 (±14.14) 55 (±5.00) 80 (±0) 4 Water 25 (±18.93) 35 (±9.57) 45 (±18.93) 65 (±5.00) (U) (±6.67)b (±8.43)b (I) 30 (±8.56)a (±8.43)a 5 Tween (±3.33)b (±6.67)b 5 Water 3.33 (±3.33)b (±6.67)b
9 Mean % Mortality x % Mortality (Day 3) x % Mortality (Day 7) Bioassay # Treatment Adults Nymphs Adults Nymphs (M) (±3.33) 10 (±4.47) 30 (±4.47)a (M) 3.33 (±3.33) (±6.67) (±9.54)a 1 Tween 3.33 (±3.33) (±3.33) 0b 1 Water 3.33 (±3.33) 0 10 (±4.47) 0b (M) 10 (±10) (±15.20) (±12.01) 100 (±0) 2 GHA Botanigard (B) 3.33 (±3.33) 40 (±7.30) 30 (±11.25) 90 (±6.83) 2 Tween 10 (±6.83) 40 (±13.66) 40 (±15.49) 80 (±7.30) 2 Water 6.67 (±4.21) (±8.43) (±9.88) (±9.88) (M) 3.33 (±3.33) 40 (±7.30) (±8.02) (±4.21) 3 Unidentified Fungus (U) 10 (±6.83) 20 (±7.30) (±9.54) (±4.21) 3 Tween 10 (±4.47) (±6.14) (±8.43) (±6.67) 3 Water (±8.02) (±8.43) (±6.14) 4 F52 (M) 25 (±5.00) 25 (±12.58) 60 (±8.16) 70 (±12.91) 4 GHA USDA (B) 30 (±12.91) 20 (±14.14) 65 (±9.57) 65 (±9.57) (I) 20 (±8.16) 40 (±14.14) 55 (±5.00) 80 (±0) 4 Water 25 (±18.93) 35 (±9.57) 45 (±18.93) 65 (±5.00) (U) (±6.67)b (±8.43)b (I) 30 (±8.56)a (±8.43)a 5 Tween (±3.33)b (±6.67)b 5 Water 3.33 (±3.33)b (±6.67)b
10 Diatomaceous earth (DE) and horticultural oil tested in conjunction with F52 (M), GHA (B) and 3581 (I) Testing for increased mortality with additives 50g/L DE, 7% oil used in treatments All other procedures as before
11 Mortality Treatment x % Mortality (Day 3) x % Mortality (Day 7) Water 5.00 (±5.00) (±12.91) DE (±8.16) (±9.57) DE/Oil (±11.54)* (±8.16) Oil (±8.16)* (±12.58) 3581 (I) (±0) (±9.57) 3581/DE 5.00 (±5.00) (±14.14) 3581/DE/Oil (±0) (±10.00) 3581/Oil (±10.00)* (±9.57) F52 (M) 5.00 (±5.00) (±5.00) F52/DE 0 (±0) (±10.00) F52/DE/Oil (±10.00) (±12.58) F52/Oil (±21.60)* (±10.00) GHA (B) 5.00 (±5.00) (±5.77) GHA/DE 0 (±0) (±15.00) GHA/DE/Oil (±8.16) (±9.57) GHA/Oil (±5.00) (±10.00)
12 Mortality Treatment x % Mortality (Day 3) x % Mortality (Day 7) Water 5.00 (±5.00) (±12.91) DE (±8.16) (±9.57) DE/Oil (±11.54)* (±8.16) Oil (±8.16)* (±12.58) 3581 (I) (±0) (±9.57) 3581/DE 5.00 (±5.00) (±14.14) 3581/DE/Oil (±0) (±10.00) 3581/Oil (±10.00)* (±9.57) F52 (M) 5.00 (±5.00) (±5.00) F52/DE 0 (±0) (±10.00) F52/DE/Oil (±10.00) (±12.58) F52/Oil (±21.60)* (±10.00) GHA (B) 5.00 (±5.00) (±5.77) GHA/DE 0 (±0) (±15.00) GHA/DE/Oil (±8.16) (±9.57) GHA/Oil (±5.00) (±10.00)
13 Use of transgenic fungi successful in other insect systems Scorpion and spider toxins Fungi engineered to express spider neuropeptides Metarhizium strains tested: Hv1a-1548, Dc1a-1548, As1a-1548, Ta1a-1548 All other procedures as before
14 Mortality Fungal Growth Treatment x % Mortality (Day 3) x % Mortality (Day 7) x Days to Growth x % dead with Growth 1548 WT 20 (±14.14) 95 (±5.00) 4 (±0.87) 40 (±8.16) As1a 10 (±5.77) 80 (±14.14) 2 (±0) 20 (±8.16) Dc1a 15 (±9.57) 70 (±10.00) 2.67 (±1.11) 40 (±8.16) Hv1a 30 (±10.00) 65 (±5.00) 3.25 (±1.43) 40 (±8.16) Ta1a 20 (±8.16) 75 (±9.57) 8 (±0) 10 (±5.77) Water 20 (±8.16) 60 (±14.14) - -
15 Overall levels of mortality low Little difference seen between wild-type strains (with and without additives) and transgenic strains Low virulence of fungi No indication that any of the entomopathogenic fungi evaluated would be effective as a biological control Weiguo Fang
16 Many insects protect themselves from fungal infection via chemical defense Earwigs Sawflies Bed bugs Pentatomids Brown marmorated stink bug defensive compounds may be the cause of poor fungal performance
17 Identify constituents of brown marmorated stink bug defensive compound Evaluate effects of defensive compounds against entomopathogenic fungi Fungal growth Spore germination
18 From literature and other studies Too many chemicals to ID, based search on a priori hypothesis Predict that trans-2-octenal and trans-2-decenal are potential candidates Analyze brown marmorated stink bug secretions to confirm presence of trans-2- octenal and trans-2-decenal AccuTOF mass spectrometer equipped with confined Direct Analysis in Real Time (cdart) ion source
19
20 3 fungi evaluated F52 (Metarhizium), GHA (Beauveria), ARSEF 3581 (Isaria) Fungi plated on PDA media 5 µl trans-2-octenal or trans-2-decenal applied to filter paper disc on inside lid of petri dish 100%, 10%, 1%, 0% concentrations
21 Inhibition of fungi Fungi grown for 3 days at 27 o C # of plates with / without fungal growth recorded
22 Trans-2-octenal 100% 10% 1% 0% Control Trans-2-decenal
23 Mean % Petri dishes without Fungal Growth (Inhibition) Trans 2 octenal Concentration Trans 2 decenal Concentration Fungus 100% 10% 1% 100% 10% 1% Control F52 (M) 100% (±0) 66% (±33.33) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) GHA (B) 100% (±0) 100% (±0) 0 (±0) 100% (±0) 66% (±33.33) 0 (±0) 0 (±0) 3581 (I) 100% (±0) 0 (±0) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) Complete inhibition of fungal growth at 100% concentration No inhibition at 1% or 0% concentrations Partial inhibition at 10% concentration for both chemicals, and all fungal strains
24 Mean % Petri dishes without Fungal Growth (Inhibition) Trans 2 octenal Concentration Trans 2 decenal Concentration Fungus 100% 10% 1% 100% 10% 1% Control F52 (M) 100% (±0) 66% (±33.33) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) GHA (B) 100% (±0) 100% (±0) 0 (±0) 100% (±0) 66% (±33.33) 0 (±0) 0 (±0) 3581 (I) 100% (±0) 0 (±0) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) Complete inhibition of fungal growth at 100% concentration No inhibition at 1% or 0% concentrations Partial inhibition at 10% concentration for both chemicals, and all fungal strains
25 Mean % Petri dishes without Fungal Growth (Inhibition) Trans 2 octenal Concentration Trans 2 decenal Concentration Fungus 100% 10% 1% 100% 10% 1% Control F52 (M) 100% (±0) 66% (±33.33) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) GHA (B) 100% (±0) 100% (±0) 0 (±0) 100% (±0) 66% (±33.33) 0 (±0) 0 (±0) 3581 (I) 100% (±0) 0 (±0) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) Complete inhibition of fungal growth at 100% concentration No inhibition at 1% or 0% concentrations Partial inhibition at 10% concentration for both chemicals, and all fungal strains
26 Mean % Petri dishes without Fungal Growth (Inhibition) Trans 2 octenal Concentration Trans 2 decenal Concentration Fungus 100% 10% 1% 100% 10% 1% Control F52 (M) 100% (±0) 66% (±33.33) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) GHA (B) 100% (±0) 100% (±0) 0 (±0) 100% (±0) 66% (±33.33) 0 (±0) 0 (±0) 3581 (I) 100% (±0) 0 (±0) 0 (±0) 100% (±0) 33% (±33.33) 0 (±0) 0 (±0) Complete inhibition of fungal growth at 100% concentration No inhibition at 1% or 0% concentrations Partial inhibition at 10% concentration for both chemicals, and all fungal strains
27 2 ml liquid PDB media containing spore suspension added to 35 mm petri dishes Trans-2-octenal and trans-2-decenal added at same concentrations as previous experiment Dishes photographed at 400x magnification 1 day after treatment, % spore germination calculated Spore germination defined as presence of germ tube
28 Germinated F52 Ungerminated F52
29 Percent Spore Germination 100% 80% 60% 40% 20% F52 (M) GHA (B) 3581 (I) 0% % Trans-2-octenal Concentration (%) Percent Spore Germination 80% 60% 40% 20% 0% F52 (M) GHA (B) 3581 (I) Trans-2-decenal Concentration (%)
30 Trans-2-octenal and trans-2-decenal present in live brown marmorated stink bug defensive secretions Both chemicals inhibit fungal growth and spore germination in laboratory setting
31 Additional challenges with using entomopathogenic fungi as biological control Low and inconsistent mortality Additives and transgenic strains do not improve efficacy Defensive chemicals may explain low virulence of fungi against brown marmorated stink bug Inhibition of fungal growth/spore germination
32 Inducing mutations in wild-type strains with the hopes of getting a beneficial mutation Attempt to induce mutation that confers resistance to stink bug defensive compounds Fewer obstacles to use than with GMO products
33
34 Thesis Committee Funding Paula Shrewsbury USDA-NIFA SCRI Award Raymond St. Leger # Joseph Patt Nancy Harding Galen Dively Lab Ray St. Leger Lab Hsiao-Ling Lu Brian Lovett Yue Li Joe Torella
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