Use of Microorganisms on Pest Management Eduardo Hidalgo, CABI Bioscience / [email protected] Alternatives and Strategies

Use of Microorganisms on Pest Management Eduardo Hidalgo, CABI Bioscience / E.Hidalgo@Cabi.Org Alternatives and Strategies

Use of microorganisms on pest management Eduardo Hidalgo, CABI Bioscience / [email protected] Alternatives and strategies

Chemicals Virus Bacteria

Crop phenology

Economics $ Instar/ Interactions Population Macro BC Epizootiology Fungi

50 agentes 45 Distribucion de huevos en cuadricula 10x10m, Lote marginata verde, Luis Jimenez 40 18 35 Numero de huevos 30 16

25 14 7

20 6 12

15 5 10 10 4

5 8 3 0 6 1 2 3 4 5 6 7 2

4 1

0 2 Aprox. 30 grados de inclinacion

0 0 2 4 6 8 10 12 14 16 18 20

Borde Distribution Applications / Efficacy Action Functional thresholds trades

Metabolites Extracts

Formulation Biotic factors controlling Diaphorina citri

Name Type Stage of D. citri controlled Tamarixia radiata Parasitoids Nymphs Diaphorencyrtus aligarhensis Coccinellids (Curinus coeruleus Mulsant, Exochomus childreni Mulsant, Harmonia axyridis Pallas, O. v-nigrum Mulsant, Cycloneda sanguinea L., and Coelophora inaequalis), Predators Nymphs chrysopids (Ceraeochrysa sp. and Chrysoperla sp.)

Syrphid flyes histerid beetle: Saprinus chalcites Carabid, Egapola crenulata Cockroach Blattella asahinai, Spiders Adults Hirsutella citriformis Adults (23 to 75% control) Isaria fumosorosea (= Paecilomyces fumosoroseus) Adults (up to 95% ctrl-Lab) Entomopathogens (57-96% ctrl-field)

Lecanicillium lecaniil, L.longisporum Adults (70% naural ctrl)

Beauveria bassiana Adults (57-96% ctrl-field)

Cladosporium sp. Adults Qureshi, J. and Stansly , P. 2020. Asian Citrus Psyllid. CABInternational 2020 Disease (a dynamic process)

Epizootic: High mortality in short time Enzootic: Low mortality all year round Factors Susceptibility Disemination

Patogenicity Interaction Virulence (DL and CL50) and TL50 Toxins Latencia Inoculum production Environment Temperature Humidity Radiation Other (soil, plant, management practices) Epizootic curve

50 600 - 600 45 Number of insects % infection 40 500 - 500

35 Economic threshold 400 30 - 400

300 infection 25 - 300 20

200 % of of % 15 - 200

10 100 - 100 5 Epizootia Pre Post

0 0 1 2 3 4 5 6 7 - 0 1 2 3 4 5 6 7 Weeks Use of entomopathogens reported in the Caribbean

Natural enemy Pest Crop Country

Aeneolamia sp. Sugar cane T&T1 2 Metarhizium anisopliae Aeneolamia flavilatera Sugar cane Guyana Citrus weevils Citrus Jamaica1 Paecilomyces fumosoroseus Bemisia tabaci Tomato T&T1 Neozygites parvispora Thrips tabaci - Barbados2 Aleurodicus cocois Virgin1 Aschersonia aleyrodis - Aleurothrixus floccosus Islands

Beauveria globulifer Trhips Cacao Grenada1

Citrus weevils Citrus Beauveria bassiana Coffee berry borer Jamaica1 Coffee Orthezia praelonga Onion, Beauveria bassiana Spodoptera exigua scallion, Jamaica1 Baculovirus formulations callaloo Trhichoderma ovalisporum Frosty pod rot Cacao Jamaica1 Coconut Simplicillium sp. Red palm mite Barbados1 palm Colletotricum musae (dead spores) Banana Crown rot Various1 Gliocladium, Pythium, Trichoderma Banana (fungal complex) countries and Lecanicillium Qureshi & Stansly1. 2020, Hajek et al., 20162 Entomopathogenic viruses

• DNA/RNA chains + protein • Obliged pathogens • Frequent in Lepidopteran larvae • Cause disease and death in few days • Can cause epizootics • Various families reported but Baculoviridae the one commonly used How to use virus for pest control

Multiplication Colonization

Ingestion

pH > 7.5 I Death Virion I Nucleous

Wind I I rain Inoculation insects Man Disemination Release Soil particles Entomopathogenic bacteria

• General information – No membrane in nucleoplasm – Reproduction by fission – Saprophytic, symbiotic, pathogenic

• Direct or potential pathogens • Spore forming / non-spore forming • Bacillus, Serratia, Pseudomonas Entomopathogenic bacteria

• Midgut bacteria Serratia entomophila S. Proteomaculans -Slow action -Larva stops feeding Bacillus thuringiesis

• Mainly for Lepidoptera but there are strains for coleopteran and mosquitos • Can be isolated from soil and infected larvae • Forms a spore and protein crystal • Can be mass produce by fermentation Bacillus thuringiensis Bacillus popilliae

Bacillus popilliae (Paenebacillus popilliae)

– Specific for Scarabaeidae – Produces a spore and parasporal bodies – Milky disease in vivo production of Bacillus popilliae

Inoculation

Extraction

Reproduction Storage Screening of Bp strains against Phyllophaga elenans L3, 16DAI, via injection.

100 Asintom. 80 Bp

40 Bp / Mort. Bp/ 20

Ctrl

228 492 495 382 359 497 393 290 292 493 283 510 504 490 285 494 496 508 Aislamiento Mortality of P. elenansL1 y L2, 35 days after inoculation with B. popilliae (292) 1x109 spores/ml

100 a a a 80 a 60 40 b

% Mortalidad % 20 0 Testigo Polvo Polvo Líquido Líquido 1A 2A 1A 2A Tratamiento L1 100

40 % Mortalidad % 20

0 Testigo Líquido 1A. Líquido 2A. Tratamiento L2 Quality control / Identification kit

•Descriptive methods :Bergey´s Manual: (Breed 1954) •Metabolism of carbohydrates (Biolog) •API (Assimilation of carbohydrates; reduction of nitrates)

•APIZYM (enzimes)

•Molecular methods Entomopathogenic fungi

Epizootics

•Agostino Bassi, 1834 Cordiceps

•>700 species/100 genus Nomurea

•Multipple environments Entomophthora

•Commercial formulations Verticillium (Lecanicillium)

Hirsutela Difficult to mass produce

Cordiceps sp.

Beauveria sp.

Widely used Life cycle of an entomopathogenic fungus (Deuteromicete)

Disemination Sporulation

Germination death

Penetration

Multiplication Toxin production Beauveria bassiana

Conidiogenous cells: • base swollen • conidia borne on denticles

Denticulate rachis is the only essential diagnostic feature defining this genus

Controls: Coleoptera-Lepidoptera Homoptera-Hemiptera Control of Cosmopolites sordidus with Beauveria bassiana

Application method: Pseudostem traps Approx dose: 109 conidia/trap Metarhizium anisopliae

Conidia: • in laterally adherent chains (forming prismatic columns or solid plates) • cylindrical (with waist) Conidiophores: • Candelabrum-like branching

Controls: Coleoptera-Lepidoptera Homoptera-Hemiptera Lecanicillium lecanii

Conidiophores bear pairs or whorls of awl-like phialids Conidia born in apical slime balls

Controls: Aphids, scale insects Whitefly, Coffee leave rust Hirsutella spp.

• Conidiogenous cell with • swollen base and long, narrow neck • Conidia usually in small slime drop or with slime coating • Synnematous

Controls: Mites, Coleoptera-Homoptera Aschersonia

•Conidia in ascas (limospores) •Attacks scale insects Paecilomyces fumosoroseus

• Phialids with distinct neck • Conidia in chains in divergent clusters • Synnematous or mononematous • Last monograph was Samson (1974) • Samson now moving to reclassify Section Isarioidea back into Isaria Controls: Hemiptera, Coleoptera, Lepidoptera P. lilacinus: Nematodos Zygomycetes

• Entomophthorales – More than 20 genus • Can cause epizootics • Presence of rhizoids and papila conidia

Controls: Diptera, Ortoptera, Homoptera, Mites What affects their efficacy?

• UV radiation • Mixing with fungicides • Alkaline pH • High temperatures • Poor quality of spraying How to produce entomopathogenic fungi

1. Isolate from insect or directly from soil using bait insects or media with antibiotics 2. Use monosporic isolates to avoid contaminants and assure genetic homogeneity 3. Inoculate the solid substrate (rice) with a conidial suspension of aprox. 1x106 con/ml, 10ml per 100g of substrate in polypropylene bags. 4. Incubate at 24-26 0C for 10 to 15 days 5. For large production, multiply the inoculum with a 3 day liquid fermentation in nutrient broth 6. Some fungi produce low amount of conidia or need a longer period of time to produce. Production of blastospores vs cost/litre using different liquid media

1.0E+07 9.0E+06 Blastospore production Blastospores 8.0E+06

7.0E+06 Conidias 6.0E+06

5.0E+06 media

4.0E+06 Conidias/ml Macro and microconidia 3.0E+06

2.0E+06 Soybean Referencemedia

1.0E+06

0.0E+00

Soya Leche Medio L Nitrato 1 Nitrato 2 Extracto L1 Extracto L2 Bayfolan 1 Bayfolan 2 Neopeptona 1 Neopeptona 2 Medio de cultivo Chlamydospores

8.0 Advantages 6.89 7.0 • Faster than solid fermentation (3-7 days) 6.0 • Works for fungi of difficult production 5.0 4.03 • Some produce better in low cost media 4.0 3.72 3.65 Costo/L ($)

3.0 2.10 Costoen $/L 2.0

1.0 0.55 0.55 0.61 0.67 0.12 0.07 Disadvantages 0.0 • Higher risk of contamination

Soya Leche • Need to adjust media accordingly Medio L Nitrato 1 Nitrato 2 Extracto L1 Extracto L2 Bayfolan 1 Bayfolan 2 NeopeptonaNeopeptona 1 2 • Some fungi do not produce blastospores Medio de cultivo Quality Control

Check for: Calculate concentration using • Concentration a haemacitometer • Viability • Virulence • Contaminants Rhizopus oryzae

• Genus/Species: Rhizopus oryzae • Slide Reference #: GK 587 http://www.doctorfungus.org • Image Type: Microscopic Morphology • Disease(s): Systemic Zygomycosis (Mucormycosis) Rhizopus oryzae

Rhizopus oryzae

• Genus/Species: Rhizopus oryzae • Slide Reference #: GK 582

http://www.doctorfungus.org • Image• Genus/Species: Type: Rhizopus oryzaeClinical •PresentationSlide Reference #: GK 583 • Disease(s): Rhinocerebral

http://www.doctorfungus.org • Image Type: Miscellaneous • Disease(s): Rhinocerebral zygomycosis zygomycosis Thank you