Mass production of entomopathogenic nematodes for plant protection
Date
2008-07-16T10:27:57Z
Authors
Nyamboli, Mirabel A
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Abstract
Several entomopathogenic nematode (EPN) species of the genera Steinernema and
Heterorhabditis are currently under evaluation for mass production and field efficacy for
biological control of insect pests. Two indigenous EPNs, namely Heterorhabditis
bacteriophora and H. zealandica, and two exotic EPNs, namely: H. indica LN2 and
Steinernema feltiae SN were examined in this study. Their symbiotic bacteria, namely:
Photorhabdus luminescens subspecies laumondii, P. luminescens, P. luminescens subspecies
arkhustii and Xenorhabdus bovienii respectively, were isolated from EPN-infected insect
host haemolymph and cultured successfully in NBTA, MacConkey agar and nutrient broth.
The growth curves, mean generation times and growth rate constants were calculated for
each of the symbiotic bacteria. X. bovienii had the smallest growth rate constant (1.99 hour-1)
while P. luminescens had the longest (2.45 hour-1). Doubling time was similar for all bacteria
(range 18 – 19.62 min). Optimum growth temperature was determined to be 28 oC for all
EPNs; except for P. luminescens isolated from H. zealandica- it had an optimum growth
temperature of 25 oC. Bioluminescence was detected in all phase I Photorhabdus species
(symbionts of the Heterorhabditid nematodes).
Three kinds of bioassays- namely: the exposure time bioassay, the on-on-one bioassay and
the dose-response bioassay were performed to determine the infectivity of the four
nematodes. In the exposure time assay, T. molitor larvae and pupae and G. mellonella larvae
were exposed to IJs of H. bacteriophora. Overall, insect mortality increased with longer
exposure times. G. mellonella larvae experienced the highest mortality. All four EPNs were examined in the one-on-one bioassay. Individual insects were exposed to single IJs of the 4
EPNs. H. indica killed 62.5% of insects after only 48 h of exposure, but H. zealandicainfected
insects experienced the highest mortality (87.5%) at the end of the 96 h of exposure.
H. indica-infected cadavers produced the highest number of IJs (195,400 IJs) while the least
(130,000 IJs) was produced by H. zealandica-infected cadavers. In the dose response
bioassay, different IJ concentrations (1, 5, 10, 25, 50, 100, 200 and 500) of the four EPNs
were applied per T. molitor larva. The bioassay was carried out in two parts. In the first part,
mortality data was collected for H. bacteriophora and H. zealandica. Significant differences
in insect mortality were apparent as early as 24 h post nematode application; the differences
were mostly between the very low and high IJ doses such as 5 and 500 IJs/insect. However,
no differences were noted when mortality data were compared between IJ doses at both 72 h
and 96 h following IJ application to the insects. In the second part of the dose response
bioassay, the number of progeny IJs emerging from EPN-infected cadavers was determined
for all four EPNs. No significant differences were noteworthy in the number of emerged IJs
at all doses for each nematode species.
The role of two factors in EPN recovery, growth and reproduction in liquid culture were
investigated, namely type of lipid and the role of EPN symbiotic bacteria. H. bacteriophora
and S. feltiae were inoculated at densities of 2500 IJs/ml into monoxenic and axenic egg yolk
liquid cultures, incubated on a platform shaker set at 25 oC and 150 rpm. Progeny IJs were
counted on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25 and 30 following IJ inoculation. H.
bacteriophora did not produce any offspring but S. feltiae recovered and produced very few
IJs in axenic culture. However, recovery, growth and reproduction were observed in
monoxenic cultures of both EPNs. The difference between IJ yield in monoxenic and axenic
cultures were highly significant from days 4 up to 30 (P < 0.05). Five millilitre aliquots of P. luminescens arkhustii were added into different H. indica egg yolk cultures, 10, 15, 20, 25
and 30 days following inoculation of 2300 IJs/ml. The cultures were incubated on a platform
shaker set at 150 rpm and 25 oC. Progeny IJs were counted on days 2, 5, 10, 15, 20, 30, 35
and 40 following inoculation of IJs in the cultures. The results showed that adding bacteria to
monoxenic cultures boosted IJ yield. IJ yield was highest on day 35, in cultures that had
received bacteria on days 20 and 25. H. bacteriophora and H. zealandica were inoculated at
a density of 4000 IJs/ml into monoxenic egg yolk cultures supplemented with either canola
oil, safflower oil or cod liver oil. Progeny IJs were counted on days 4, 10, 12, 14, 16, 18, 20,
22, 25, 30 and 35 following IJ inoculation. IJ yield increased constantly in cod liver oilsupplemented
cultures, but IJ yield was highest in cultures supplemented with canola oil for
both nematodes. Overall, H. bacteriophora cultures had higher IJ yields.
The consequence of desiccation on the reproduction of all four nematode species was
determined by the number of IJs emerging from EPN-infected host cadavers at different
levels of relative humidity (RH). Thirteen different humidity environments with levels
ranging from 70.4 – 100% RH were prepared using glycerol and water. EPN-infected T.
molitor larvae were exposed to any of the 13 RH levels, but the cadavers were
preconditioned at a higher RH. More precisely, preconditioning occurred at the RH preceding
that for which it was intended, for 24 h before being exposed to the intended lower RH.
Cadavers were randomly selected from all 13 RH environments and placed on White traps
and progeny IJs counted, for all four nematode species. H. indica-infected cadavers had the
highest IJ yield, except at RH ≥ 97.8% where S. feltiae-infected cadavers produced more IJs.
However, the number of IJs produced by S. feltiae drastically reduced at lower RH levels ≤
85.3. The number of IJs produced per host infected by either H. bacteriophora or H.
zealandica was similar across all RH levels. The relationship between RH treatment and the number of IJs produced per cadaver was linear P < 0.001 for all EPNS; r2 = 0.97, 0.94, 0.94
and 0.90 for H. indica, H. bacteriophora, H. zealandica, and S. feltiae respectively. Overall,
IJ yield was significantly different between very high (≥ 97.8%) and very low RH levels (≤
77.2%).
The feasibility of formulating EPN-infected T. molitor larvae to overcome the hindrance of
handling and storage was tested using H. indica. Firstly, T. molitor was cultured in bran. The
different bran cultures had variable numbers of adults ranging from 10 to 100 (in increments
of 10), with a male/female sex ratio of 50:50. Half of the cultures were sieved to separate the
eggs from the adults, but other cultures were not sieved. The results indicated that sieving
was useful in improving T. molitor yield. Also, it was perceptible that cultures with too many
(≥ 80) or too few (≤ 30) adults per container produced fewer offspring in both cultures that
were sieved and those that were not. Secondly, 12 combinations of dipping and rolling agents
were tested for formulating 4 and 8 day old H. indica-infected T. molitor larvae. The dipping
agent coated the surface of the cadaver and served as an adhesive for the powder, which was
intended to prevent cadavers from sticking together. Three of the formulations failed to
adhere to either 4- or 8-day-old cadavers. The remaining 9 formulations were tested for their
effects on nematode reproduction. Cadavers were formulated at 4 days post inoculation (PI)
and 8 days PI and placed in White traps. There were no progeny IJs from 5 of the
formulations. Of the remaining 4, the most promising formulation was the 2.5MC because
the cadavers coated with the formulation produced IJs that were significantly higher in yield
than other formulations for both 4 and 8 day old cadavers. The other three successful
formulations were 2.5SC, 1SC and 1MS.