Formulation Storage and Quality

The important aspects, which are to be kept in mind for commercialization of entomopathogenic nematodes as biocontrol agent are formulation, storage and quality control. Formulation refers to the preparation of a product from an ingredient by the addition of certain active (functional) and non-active (inert) substances. It provides means to improve the activity, delivery, ease to use, storage stability and field efficacy of the nematodes. Entomopathogenic nematode species have differential requirement for temperature, moisture and oxygen (Glazer 2002). These requirements may dictate the conditions for formulation and storage. As a result of varied nematode species, differential survival requirements and formulation types, an array of products can be developed for management of different insect pests. Entomopathogenic nematodes are live organisms and regardless of how they are formulated, their quality declines with time. Furthermore, all formulations are susceptible to temperature extremes, ultraviolet light, anoxic conditions and contamination (Lewis and Perez 2004). Infective juveniles of entomopathogenic nematode can be stored in water for several months in refrigerated bubbled tanks, however, high cost as well as quality maintenance are somewhat difficult through this method. Tolerance and activity of the nematodes at extreme environmental conditions can limit the shelf life, quality and field performance of the products (Ehlers et al. 2005). Till now no entomopathogenic nematode formulation has met the 2-year shelf life requirement of a standard chemical pesticide (Table 13.4).

The target in developing an ideal formulation is (i) maintenance of quality, (ii) increased storage stability, (iii) low transport cost, and (iv) enhancement of nematode survival during and after application. These can be achieved when absorbents, adsorbents, anticaking agents, antimicrobial agents, antioxidants, surfactants, carriers, preservatives, ultra violet protectants, etc. may be added to the formulation depending upon the need. Formulation of nematodes for storage and transport are generally done by two ways.

1. The nematodes are placed in inert carriers such as sponge and vermiculite that allow free gas exchange and movement of nematodes.

2. Addition of functional ingredients, which reduces nematode activity and metabolism.

It has been observed that sometimes nematodes escape from the inert carriers and dry out (Grewal and Peters 2005), therefore in formulations mobility/metabolism of nematodes is minimized through physical trapping, inclusion of metabolic inhibitors or through the induction of partial anhydrobiosis. Nematode metabolism is temperature driven and a warm temperature between 20°C and 30°C accelerates metabolic activities, thereby reducing nematode viability (Georgis 1990a). Formulations prepared in carriers such as alginate, clay, polyacrylamide gels, ver-miculite, activated charcoal, etc. can be stored for at least 3 months under refrigeration or at room temperature. Temperature requirement during storage, however,

Table 13.4 Expected shelf life of different entomopathogenic nematode formulations

Formulation

Nematode species

Strain

Shelf life (months)

22-25°C

2-10°C

Sponge

Steinernema

All

0.03-0.01

2.0-3.0

carpocapsae

Heterorhabditis

HP88

0

1.0-2.0

bacteriophora

Hybrid

0

0.75-1.5

H. indica

LN2

0.25

0

H. marelata

Oregon

0

1.0-2.0

Vermiculite

S. carpocapsae

All

0.1-0.2

5.0-6.0

S. feltiae

SN

0.03-0.1

4.0-5.0

Liquid concentrate

S. carpocapsae

All

0.16-0.2

0.4-0.5

S. riobrave

RGV

0.1-0.13

0.23-0.3

Wettable powder

S. carpocapsae

All

2.0-3.5

6.0-8.0

S. feltiae

UK

2.5-3.0

5.0-6.0

ENO2

0.5-1.0

3.0-4.0

S. glaseri

NJ43

0.03-0.06

1.0-1.5

S. scapterisci

Uruguay

1.0-1.5

3.0-4.0

H. bacteriophora

ENO1

0.5-1.0

2.0-3.0

H. indica

LN2

0.25-0.50

0

H. megidis

UK

2.0-3.0

4.0-5.0

H. zealandica

X1

1.0-2.0

0

Water-dispersible granule

S. carpocapsae

All

4.0-5.0

9.0-12.0

S. feltiae

SN

1.5-2.0

5.0-7.0

S. riobrave

RGV

2.0-3.0

4.0-5.0

Alignate gel

S. carpocapsae

All

3.0-4.0

6.0-9.0

S. feltiae

SN

0.5

4.0-5.0

Flowable gel

S. carpocapsae

All

1.0-1.5

3.0-4.0

S. glaseri

NJ43

0.16-0.2

0.4-0.5

S. scapterisci

Colon

0.1-0.13

0.23-0.3

varies with entomopathogenic nematode species. General range of storage temperature for steinernematids is 5-10°C, whereas for heterorhabditis it is 10-15°C (Georgis 1990b). In another approach functional ingredients such as alginate and flowable gel formulations are used to trap nematodes physically in order to reduce their movement. Also with the induction of partial anhydrobiosis, nematode activity and metabolism can be reduced. Grewal (2002) reported the storage of S. car-pocapsae for 3-4 months at 25°C and S. feltiae for 2-4 weeks in alginate gel formulation. Bedding (1988) described a formulation whereby nematodes were mixed in clay for removing excess surface moisture and inducing partial anhydro-biosis. The formulation called 'sandwich' consisted a layer of nematode between two layers of clay.

Water-dispersible granule formulation is considered to be the first commercial formulation enabling storage of S. carpocapsae for 6 months at 25°C at a concentration of over 300,000 infective juveniles per gram (Grewal 2000). When stored at

Table 13.5 Formulations of Steinernema and Heterorhabditis developed by different countries

Nematode

Product

Country

Steinernema carpocapsae

Ortho biosafe

United States of America

Bio vector

United States of America

Exhibit

United States of America

Sanoplant

Switzerland

Boden nutzlinge

Germany

Helix

Canada

S. feltiae

Manget

United States of America

Nemasys

United Kingdom

Stealth

United Kingdom

S. riobrave

Vector MG

United States of America

S. scapterisci

Proactant Sc

United States of America

S. kushidai

SDS biotech

Japan

Heterorhabditis megidis

Nemasys

United kingdom

H. bacteriophora

Otinem

United States of America

E- Nema Gmbh

Germany

room temperature, water-dispersible granule formulations were found prone to microbial contamination. Therefore, antimicrobial and antifungal agents are often added to suppress the growth of these microbes.

Application of nematodes in infected insect cadavers have also been described by some workers (Shapiro-Ilan et al. 2001, 2003), which enables the slow release of nematode and therefore considered effective for small-scale application. Coating the cadavers with starch and clay mixture helps in preventing rupture during storage and shipping (Shapiro-Ilan et al. 2001).

Quality is measured in terms of degree of excellence of a product and quality control is a system of maintaining standards in manufactured products. According to Grewal and Peters (2005) quality of entomopathogenic nematode involves correct identity of species, total number of live nematodes, ratio of live and dead nematodes, matching of host finding behaviour to the target pest, pathogenicity and reproduction ability of nematodes in the target pest, age of the nematodes used, storability, heat tolerance and cold or warm temperature activity. Size and packaging, reliable instructions for the consumers, ease at transportation, absence of contaminants, product cost, availability and field efficacy are the other parameters required for the product quality (Grewal and Peters 2005). Some commercial products of entomopathogenic nematodes prepared in different countries are enlisted (Table 13.5).

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