Development and virulence of Heterorhabditis spp. strains associated with different Xenorhabdus luminescens isolates

JOURNAL

OF INVERTEBRATE

PATHOLOGY

58, 27-32 (1991)

Development and Virulence of Heterorhabditis spp. Strains Associated with Different Xenorhabdus luminescens Isolates RICHOU *Guangdong

Entomological

HAN,*

W. M. Woum,i’

Institute, Guangzhou, Research Center, Private

AND LIVING

LI*

China, and TEntomology Division, Bag, Auckland, New Zealand

DSIR,

Mt.

Albert

Received May 1, 1990; accepted September 11, 1990 axenic Heterorhabditis strains were mutually recombined with their Xenorhabdus lumisymbionts. Each strain developed successfully only on some X. luminescens isolates. Those that developed successfully were maintained on Bedding’s sponge medium and on Galleria mellonella larvae. On different X. luminescens isolates they grew at different rates and sometimes they grew more prolifically on others than on their own symbionts. The virulence of seven natural Heterorhabditis strains to Galleria larvae varies considerably and is unrelated with their growth rates and yields. 0 1991Academic PWS. h. KEY WORDS: Nematodes, parasitic; Heterorhaditis heliothidis; Heterorhabditis megidis; undescribed Heterorhabditis spp. ; bacteria; Xenorhabdus luminescens; insects; Galleria mellonella; dosage/mortality; infectivity: LD,,, LD,. bacterial retention; symbiosis; mass production. Seven nescens

tured with the symbionts of some other Steinernema spp. and even with bacteria other than Xenorhabdus spp., although they are not able to maintain nonXenorhabdus within the intestine of the infectives and are limited in their ability to maintain the symbiont of another Steinernema sp. (Poinar and Thomas, 1966; Akhurst, 1983; Boemare et al., 1983; Dunphy et al., 1985). The association between Heterorhabditis spp. and X. luminescens is much more strict (Han et al., 1990a,b). Seven axenic Heterorhabditis spp. strains (see Table 1) were combined with each other’s original X. luminescens symbiont. Most strains, to a degree, developed successfully on four or five of the X. luminescens isolates to which they were exposed. None of the strains developed on all isolates tested. On the basis of Heterorhabditis specificity, X. luminescens isolates can be distinguished into four groups. Percentage of infection containing bacteria, for each nematode strain tested, varied with different bacterial groups (Han et al. 1990b). The present paper further reports the development and virulence of the resulting combinations.

INTRODUCTION

Entomogenous nematodes of the families Heterorhabditidae and Steinernematidae are obligate parasites of insects and are potential biological control agents for insect pests. The infectives of these nematodes symbiotically maintain the bacteria of the genus Xenorhabdus (family Enterobacteriaceae) in their intestines. The symbiotic association between them plays an important part in the reproduction and pathogenicity of this group of nematodes. The nematodes provide protection for the bacteria outside the insect host and a tool of transmission between the insect host. The nematodes also secrete an inhibitor for some host defense mechanisms. The bacteria contribute to the association by causing septicemia followed by death of the insect, by producing antimicrobial substances to protect the infected cadaver against invasion by other microorganisms, and by providing nutrients for nematode development and reproduction. Monoxenic in vitro culture methods for mass reproduction of this group of nematodes have been established based on the presence of their bacterial symbionts Xenorhabdus spp. (Bedding, 1981; Wouts, 1981). Most of Steinernema spp. can be cul-

MATERIALS AND METHODS Bacteria. Heterorhabditis populations 27 0022-201 l/91 $1.50 Copyright 0 1991 by Academic F’ress. Inc. AU rights of reproduction in any form reserved.

28

HAN, WOUTS,

AND LI

TABLE 1 SOURCESOF NEMATODES AND BACTERIA Bacterium

Nematode host strain

Origin

Heterorhabditis Xenorhabdus X. luminescens X. luminescens X. luminescens X. luminescens

HO6 (XlHO6) H3 (XlH3) G12 (XIG12) Q6 (XlQ6) V16 (XlV16)

luminescens

sp. sp. sp. sp. sp.

HO6 H3 G12

46 V16

J. Wang, Shandong, China Y. Wang, Hailing, China R. Han, Hainan, China J. Liu, Fujian, China R. A. Bedding, CSIRO, Australia

H. megidis X. luminescens

HNA (XlHNA)

X. luminescens

HNZ (XIHNZ)

sp. HNA

T. Jackson, MAFTech,

New Zealand

H. heliothidis

sp. HNZ

from which bacterial strains of X. lumineSceltS were isolated are listed in Table 1. The isolates were obtained from insect blood from the hemocoel of Gulleria mellonella larvae infected 48 hr with infective Heterorhabditis juveniles. Bacteria were cultured and tested at 20°C. Stock cultures were maintained on NBTA (nutrient agar plates with 0.0025% (w/v) bromothymol blue and 0.004% (w/v) triphenyltetrazolium chloride; Akhurst, 1980) at 12°C and subcultured monthly. Mass production of successful nematodelbacterium associations in sponge culture and in Galleria larvae. Monoxenic cul-

tures of nematode/bacterium combinations were established by Han et al. (1990b) on fortified lipid agar (1.6% nutrient broth, 1.2% agar, and 1% corn oil; Wouts, 1981). The combination of a Heterorhabditis population with a Xenorhabdus isolate was considered a successful nematode/ bacterium association if nematode development continued after three biweekly subcultures onto new bacterial lawns of the same Xenorhabdus isolate on fortified lipid agar. When a nematode/bacterium combination did not develop successfully, at least four repeat attempts were made. Successful nematode/bacterium associations were reared in sponge cultures (Bed-

W. Wouts, DSIR, New Zealand

ding,

1981; Wouts, 1981) and in G. mellonella larvae (because Heterorhabditis sp. HO6 from XlHNA lawns failed to reproduce in infected larvae, infectives of that population were injected into the insect larvae with XlHNA bacteria). The sponge culture medium consisted of 1% peptone, 1% beef extract, 20% eggs, 15% soya flour, 5% corn oil (by weight), 8% polyether polyurethane sponge, and 50% distilled water (modified from Li et al., 1987). An 8-g sponge culture medium was placed in 50-ml conical flasks and autoclaved. Each flask was then inoculated with 2 ml of a 24-hr-old primary form bacterial suspension in peptone-salt water, incubated for 2 days, and inoculated with five monoxenically grown gravid females at 25°C. Average yields were estimated after 3 weeks by counting extracted infectives in two replicate flasks. Yields were ranked as +++++, ++++, +++, ++,or + for2.8 x 106-2 x 107, 2 x 106-2.8 x 106, 1.4 x 106-2 X 106, 8 X 105-1.4 x 106, or <8 x IO5 infective juveniles per flask, respectively. For mass production in G. mellonella larvae, sixth instars weighing 179 + 7.8 mg were infected by exposure to infective juveniles from fortified lipid agar cultures, on damp filter paper. Developing infectives were extracted from the cadaver on a White water trap.

VIRULENCE

OF

SEVEN

Bioassay. Of each successful nematode/ bacterium combination, after two serial subcultures through G. mellonella larvae, 7 + 2-day-old infectives were bioassayed: 0, 2’, 2l, 22, 23, and 24 infectives, in 1 ml of 0.1% formalin solution, were placed on 3 cm sterile tine sand, and moisture content approximately 6% covering a Galleria larva in a glass scintillation vial (diameter = 2.5 cm, height = 4.5 cm). The vials were loosely closed with the screwcaps. Nematode levels were established by counting, using a dissecting microscope. There were 20 replicates at each dose (number of infectives) in each treatment combination. Larval mortality was assessed after 96 hr at 20°C. The color and luminescence of infected larvae were determined and the symbionts were reisolated. Because of the large number of combinations involved, bioassays were done in three sessions. The natural Heterorhabditis sp. HO6 combination was included as a standard in each session. The probit analysis computer program, POLO (Russell et al., 1977), was used to determine dosage/mortality lines for each

combination. The virulence of each combination relative to the strain HO6 was calculated as described by Finney (1971). RESULTS Monoxenic cultures. Yields obtained from sponge cultures are presented in Table 2. In the different bacterial cultures the nematode developed at different rates and sometimes more prolifically on others than on their own symbionts. Strain H3 and G12 showed the best potential for nematode reproduction in sponge medium. There was a delay of development in XlHNA + HNA (for combination symbols see Table 3), with many females still present after 3 weeks. Bioassay. As an example of the data, the three HO6 sets of results gave responses (number of dead Galleria larvae out of 20; at doses 2’, 2’, 22, 23, and 24 infectives) 0,2,6,11,17,20; 0,5,6,13,17,18; 0,3,5,11,16,19. The LD,,, LD,, and relative potencies, with 95% confidence limits for the tested combinations, are presented in Table 3. Adjustment for heterogeneity in the data was

TABLE YIELD@

Nematode strain H. megidis Heterorhabditis

sp. HO6 sp. G12 sp. H3

OF INFECTIVES

29

Heterorhabditis

2

IN IN VITRO CULTURES OF NEMATODE/BACTERIUM FOR 3 WEEKS AT 25°C

COMEIINATIONS

Combinations maintained in sponge culture Bacterium XlHNA

XlH06

XlG12

X1H3

-c

-

-

+++ ++++ +++

+++ + n

-e +++++

++++ ++++

n

+++

-

n n

n n

-

wkb

XIHNZ

XIQ6

XIV16

n

n

++ -

n -

n -

-

++

n

n

-

n n

n n

n n

nd

H. heliothidis

HNZ Heterorhabditis

46 VI6

a + + + + +, 2.8 x 106-2 x IO’; + + + +, 2 x 106-2.8 x 106; + + +, 1.4 x 106-2 x 106; + +, 8 x IOL1.4 x 106; +, <8 x 105. b There was a delay of development in this combination with many females still present after 3 weeks. ’ Not successful combination. d Successful combination not tested in sponge culture. e There were contaminants in this combination that made no exact yield data of infectives available.

30

HAN,

WOUTS,

TABLE INFECTIVITY

Combination

COMBINATIONS

symbol

Bacterium Nematode XlHNA XIHNZ XIH06 XIHNZ XlH06 XlQ6 XlV16 XlH3 XlH3 XlG12 XlG12 XlHNA XlHNA XlH06 XlHNA XlHO6 XIHNA

OF VARIOUS

+ + + + +

HNA HNZ HNZ HO6 HO6

+ Q6 + + + + + + + + + + +

V16 G12 H3 H3 G12 H3 G12 G12 HO6 H3 HNZ

Potency0 8.34 1.91 1.27 1.05 1 0.95 0.50 0.35 0.28 0.17 0.24 0.15 0.13 0.02 d d d

AND

3

OF Galleria

95% limits 4.30-16.98 1.13-3.30 0.76-2.13 0.62-t .75 P 0.57-l 59 0.30-0.83 0.2W.58 0.16-0.47 O.l(M.30 0.14-0.41 0.08-0.26 0.07-0.23 0.01-0.06 d d d

LI

mellonella

L&o 0.2 2 2 3 3 3 6 13 14 14 19 27 80 d d d

IN SAND AT

95% limits b

l-2 l-3 2-t 2-I 2-6 4-9 7-68 8-55 IO-29 10-147 14-351 b < d d d

20°C FOR 96 hr

L&o __1.9 5 17 12 9 12 27 187 134 51 220 224 1873 c d d d

95% limits -__ b

4-9 9-72 8-25 7-17 7-66 16-83 4443,164 40-6,954 26-278 5 l-62,484 5486,539 b

L1The potencies with 95% limits relative to the pote ICY of XlHO6 + H06. b No confidence limits computed because g 2 0.5 at 90% level. c Test suspended because less than 10% kill was achieved at highest dose level applied. d No mortality revealed. ’ XlH06 + HO6 was regarded as having a standard potency = 1.

not required. There were no significant differences among the results obtained with the control strain HO6 at the three sessions of the bioassay (equality: P = 0.282) and there was no mortality in any of the nematode-free vials. XlH06 + G12 showed mortality (2 out of 20) only at the highest dose. Comparisons of the regression lines of the seven natural combinations showed neither parallelism nor equality in response (equality: P = 0.000; parallelism: P = 0.010). The virulence of H. megidis HNA (Poinar et al., 1987) was consistently greater than that of the other strains. Of the natural combinations, Heterorhabditis sp. G12 was the least virulent. Combinations XlHNA + H06, XlHNA + HNZ, and XlH06 + H3 caused no mortality. The infected Galleria larvae always showed the pigment and luminescence characters of the symbiont present. Where (in Table 3) the relative potency was less than 0.5, the range of dose did not

extend as high as was desirable for accurate estimation of the LD5, and LD,, limits. DISCUSSION Heterorhabditis growth rates and yields in sponge culture were influenced by the bacterial isolates to which they are exposed. The best nutrient conditions for nematode growth are not necessarily established by its indigenous symbiont . This may reflect a difference in specific nutrients transformed by different bacterial isolates. Both H3 on XlG12 cultures and HNZ on XlHO6 cultures were more vigorous than on their indigenous symbionts. The seven natural nematode strains grew at different rates with HNA developing far slower than H3 and G12, which indicates the importance of choosing a nematode strain with high growth rates and yields from those

VIRULENCE

31

OF SEVEN Heterorhabditis

nematode strains with equal effectiveness as possible control agents for a particular insect. The symbiotic bacteria support their associated nematodes by converting a wide range of substrates into media for the reproduction of the nematodes. However, how these bacteria establish suitable conditions for nematode reproduction needs to be researched further. The virulence of natural strains to Galleria larvae varies considerably, which confirms the inter- and intraspecific variation in the host infectivity of steinernematid and heterorhabditid nematodes reported by Bedding et al. (1983) and Molyneux et al. (1983). Strain HNA produces greater Galleriu mortality than the other strains tested. Against grass grub (Costelytra zealandica (White)). however, strain HNA is less infective than V16 and HNZ (Wright et al., 1988). This discrepancy may reflect a difference in host insects and assay conditions (e.g., texture and moisture of sand). Since strains H3 and G12 reproduce well in sponge media but do not show great virulence against greater waxmoth larvae, Heterorhabditis nematode growth rates and yields are considered unrelated to their virulence. In the successfully developing combinations, the percentage of infectives containing bacteria varied (Han et al., 1990b). The combinations in which the infectives retained bacteria generally showed increasing virulence with an increasing number of infectives in that population retaining bacteria. Although several strains grew well on XlHNA, they did not efficiently carry XlHNA (Han et al., 1990b), which was reflected in reduced Galleria mortality. As the association between nematode and bacterial symbionts is not completely specific, information is needed when evaluating a new nematode-bacterium combination for its effectiveness against an insect pest, both on its developmental ability in artificial media and its infectivity in the field.

ACKNOWLEDGMENTS This work was undertaken at the Entomology Division, DSIR, New Zealand, as part of the China/New Zealand Scientific Exchange Programme. We thank Mr. J. F. Longworth, Director of the Entomology Division, for establishing the contacts that made this work possible and Dr. P. J. Wigley and Miss Zhu Junsheng for their advice on bioassays.

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