In Ecuador, entomogenous nematodes have considerable potential in managing soil pests, especially

Phyllophaga sp. in perennial crops such as blackberry (Rubus glaucus). Wide adoption of beneficial

nematodes by farmers is currently challenged by the need for economically feasible, local, artisanal mass

production of efficacious nematode species/strains.

Nine strains of Steinernema and Heterorhabditis nematodes from the lNlAP collection, were evaluated

greenhouse tests using blackberry plants infested with Phyllophaga sp. The best nematode was

Heterorhabditis bacteriophora strain H-04D. It caused 70% larvae mortality and increased production of

blackberry roots (10.8 gram roots per plant versus 2.7 grams in the untreated controls).

Artisanal production of this nematode used compost, into which 100 larva Phyllophaga sp. from farmers’

fields, were placed. Then 15 larval Galleria mellonela larvae infected with the nematode were added.

Infective juveniles (IJs), were found up to 90 days after start of the test production. The nematode population

that can be obtained by this method depends on the number of larvae of Phyllophaga sp. used at the

beginning of the preparation of the compost. One larva can produce up to 32,000 IJs. In addition, the farmer

will be able to establish composting and multiply more nematodes from the inoculum available. Soil and

compost from the nematode can be used in crops for the control of pest mentioned.

ABSTRACT

INTRODUCTION

METHODOLOGYA. SELECTION OF THE BEST ENTOMOPATHOGENIC NEMATODE STRAIN

REFERENCESCastillo, C.; Gallegos, P.; Asaquibay, C.; Oña, M. eds. 2012. Guìa de prospección y producción de nematodos entomopatógenos. INIAP, EESC, Departamento Nacional de Protección Vegetal. Quito, Ecuador. Manual Técnico Nº 88. 15 p.INIAP (Instituto Nacional Autónomo de Investigaciones Agropecuarias). Departamento Nacional de Protección Vegetal. 2012. Informe Anual 2012. Estudio de la acción de los insectos de suelo en la marchitez descendente de la mora (Rubus glaucus) en la provincia de Tungurahua. Cutuglagua, Ecuador.Lemma Ebssa.; Eugene M. Fuzy.; Matthew W. Bickerton.; Albrecht M. Koppenhöfer. 2012. Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species, Biocontrol Science and Technology, 22:1, 117-123, DOI: 10.1080/09583157.2011.643767Ricci, M.; Glazer, I.; Campebll, J.F.; Gaugler, R. 1996. Comparison of bioassays to measure virulence of different entomopathogenic nematodes, Biocontrol Science and Technology, 6:2, 235 – 246 DOI: 10.1080/09583159650039421 CONTACT

Francisco Báez: francisco.baez@iniap.gob.ec, Patricio Gallegos: patricio.gallegos@iniap.gob.ec

NATIONAL AUTONOMOUS INSTITUTE FOR AGRICULTURAL RESEARCH (INIAP) - ECUADOR

Francisco Báez, Patricio Gallegos, César Asaquibay, Marcía Oña

Artisanal Multiplication of Entomopathogenic Nematodes in Ecuador for the control of Phyllophaga sp.

Larval Phyllophaga sp. are important in perennial Ecuadorean crops such as blackberry (Rubus glaucus).

Blackberry is a highly attractive fruit for the Ecuadorean, and due to its low production area, it is not

exported in large volumes but consumed domestically. The Department of Plant Protection of lNlAP, reported

a reduction in the root area of the plant, with fewer and shorter branching, causing decreased fruit yield.

(INlAP 2012).In Ecuador, the limiting factor in wide-scale use of beneficial nematodes

(EPN) is the difficulty of mass production and subsequent distribution to

farmers. Currently, these nematodes are produced in the laboratory, by

methods difficult for Ecuadorean farmers. The method requires extraction of

nematodes from larvae of Galleria mellonella by White trapping (Castillo et

al., 2012). Subsequently, these nematodes are distributed for application to

control larvae of soil, especially Phyllophaga sp. A basic artisanal production

method is needed if nematodes are to have greater adoption.

1. Efficacy tests were conducted with 2-month-old blackberry plants placed in 1.5 L plastic pots.

2. Field collected five larval Phyllophaga sp. collected from field (1.0 to 2.0 g each) were placed in each pot

and maintained for 7 days.

3. For the test were used two plastic pots per treatment more a untreated control.

4. The pots were inoculated with one of 9 EPN nine strains (Table 1), at a ratio of ten infective juveniles (IJs)

per gram of soil. (The IJs used in this study were extracted from larvae of Phyllophaga sp., previously

inoculated in the laboratory).

B. ARTISANAL MULTIPLICATION OF EPN1. Compost beds, 2.5 m long and 1.25 m wide, were established with wooden boards to retain the material.

2. Compost was a mix of local materials: one part organic matter (droppings of small animals, chickens, or

cattle) with three parts soil). All organic material used in this study was completely decomposed.

3. Introduction of 100 Phyllophaga sp. larvae and 15 G. mellonela larvae, infected with EPN.

4. Compost was protected with mulch to prevent sunlight, low temperatures, and to maintain humidity.

5. Compost was moistened weekly to keep moisture close to water field capacity.

6. Subsequent, satellite production could be made by mixing 15 kg of composted material infested with EPN

(or 15 infected Phyllophaga larvae) with the contents of the new compost along with 100 healthy larvae of

Phyllophaga sp.RESULTS AND CONCLUSIONS

We determined the best strain to be Heterorhabditis bacteriophora HO4-D in the greenhouse tests. It resulted in

70% larval mortality under the condition of the test (Figure 1) and significantly increased blackberry plant root

mass (10.8 grams ) compared with the control treatment (2.7 grams per plant) (Figure 2).

.B. ARTISANAL MULTIPLICATION OF EPN

Artisanal production of EPN is possible using Phyllophaga sp. larvae, in compost. On average, the mortality of the larvae in

compost was 78.3% and the population of IJs / larva reached a maximum of 32,000 individuals, as determined by the

laboratory extraction method (White trap). At the end of the study, on day 90 after the trial was begun, the floor of the

compost was sampled and we found an average density of 25 IJs / gram of soil. The source of organic material for the

production of compost can be chicken (Gallus gallus domesticus), guinea pig (Cavia porcellus) and rabbit (Oryctolagus

cuniculus), all of which are commonly reared on Ecuadorean farms. The compost method can be easily adopted by Ecuadorean

farmers.

Prior to development of artisanal multiplication, an efficacious nematode

strain had to be identified through efficacy tests evaluating 9 EPN from

lNlAP for the control of larval Phyllophaga sp. These trials were conducted in

blackberry plants through deliberate infestations of the pest. A compost

based artisanal production system was then devised and evaluated with the

most efficacious EPN.

 

A. SELECTION OF THE BEST STRAIN NEMATODE

Establishment of the compost bed Placement of EPN-infected larval Phyllophaga sp. and G. mellonella in the compost.

Preparation of compost by manual mixing

Figure 1. Mortality of larvae of Phyllophaga sp. after exposure to nine strains of nematodes. Error bars represent S.E.M.

Figure 2. Weight of root blackberry plants infected with larvae of Phyllophaga sp. Error bars represent S.E.M.

Collection Region Strain Designation SpeciesChimborazo – La Delicia H04 - D Heterorhabditis bacteriophoraTungurahua – Huachi 2-TH Heterorhabditis bacteriophoraCarchi – Monteverde CB-13 Steinernema feltiaeChimborazo – Guayllabamba H01-G Steinernema feltiaeCotopaxi – Chambapongo CT-08 Steinernema feltiaeCarchi – Chután Bajo CH-06 Steinernema feltiaeCotopaxi – Pataín Norte CT-13 Heterorhabditis bacteriophoraChimborazo – Rayoloma H03-R Steinernema feltiaeCotopaxi – Chanchaló CT-06 Steinernema feltiae

Table 1. EPN evaluated in the selection of the best entomopathogenic nematodes strain.

5. The test was maintained for 14 days after inoculation with EPN.

6. Larval mortality and root weight of each blackberry plant was evaluated.

7. Mortality of larvae and weight of root blackberry plants were analyzed using LSD FISHER comparison

method (InfoStat I/S Version 2010).

Blackberry plant root affected by Phyllphoga sp.

Nematode-infected Phyllphoga sp. larva (left) and healthy larva (right)