FRUITGROSCIENCE

Entomopathogenic Nematodes
show excellent potential to control soil stages of false codling moth and can
use the adult moth for aerial transport over long distances
Antoinette P. Malan1 & Sean D. Moore2

Department of Conservation Ecology & Entomology, Faculty of AgriSciences, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
Citrus Research International, PO Box 20285, Humewood 6013, South Africa

1
2

Adapted from: Malan, A.P., Knoetze, R. & Moore, S.D. (2011).

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential
against false codling moth. Journal of Invertebrate Pathology, 108:115 - 125
EPNs of the families Steinernematidae and
Summary A survey was conducted to deHeterorhabditidae are widely distributed in
termine the diversity and frequency of natusoils throughout the world. They are oblirally occurring entomopathogenic nematodes
gate parasites of insects, killing them within
(EPNs) in citrus orchards in three provinces of
48 hours after infection, with the aid of their
South Africa. The main aim of the survey was
associated bacterial symbiont. Since the late
to obtain nematodes to use as biological con1970s, these nematodes have gained status
trol agents against false codling moth (FCM),
as one of the best non-chemical alternatives
Thaumatotibia leucotreta, a key pest of citrus in
for the control of insect pests, mainly due to
South Africa. Six species of nematodes were
their ability to reach insects in cryptic habiidentified from 35 positive soil samples. In
tats, their high reproductive ability, the ease
laboratory bioassays, the six nematode speof mass producing them, and their safety for
cies found during the survey appeared to be
humans and other vertebrates. Whilst the
highly virulent against the last instar of FCM
Fig. 1. Soil stages of FCM targeted by
free-living larva or infective juvenile (IJ) is
larvae, while pupae were found to be less susEPNs.
between 0.50.9 mm long, it can be applied
ceptible. Steinernema yirgalemense, at a concento the soil with conventional spray equipment or through the
tration of 50 infective juveniles (IJs)/FCM larva caused 100% morirrigation system. Nematodes can also be used against abovetality and 74% at a concentration of 200 IJs/FCM pupa. In a sand
ground pests such as codling moth.
bioassay, S. yirgalemense gave 93% control of cocooned pupae and
In South Africa, the citrus industry currently mainly employs
emerging moths at a concentration of 20 IJ/cm2. This is the first
a combination of orchard sanitation, mating disruption and bioreport on the potential use of nematodes to control soil-borne life
logical control, using a Cryptophlebia leucotreta granulovirus, as
stages of FCM, which include larvae, pupae and emerging moths.
well as the sterile insect technique (Hofmeyer et al., 2005; Moore
It was also shown for the first time that emerging moths were in& Kirkman, 2008) to control FCM. However, none of the control
fected with nematodes, which might aid in their control and the
measures targets the soil-borne stages of FCM. As soil is the natdispersal of nematodes over long distances.
ural habitat of EPN, the last-instar FCM larvae which fall onto
Introduction The false codling moth (FCM), Thaumatotibia leuthe soil, as well as the pre-pupae, pupae and emerging moths,
cotreta (Meyrick), is endemic to South Africa and occurs in suboffer a window of opportunity for the use of nematodes as bioSaharan Africa and on the Indian Ocean islands (Newton, 1998).
logical control agents against FCM. Nematodes can also fill an
The life cycle of FCM is 2560 days, and up to six generations
important niche in early spring, summer, autumn, and after-harper year have been recorded in South Africa. The moths lay their
vest, when, traditionally, no control measures are implemented.
eggs on the fruit. The hatched larvae then penetrate the fruit,
Surveys are currently being conducted in many countries
causing premature fruit drop. The last-instar FCM larvae drop
other than South Africa to find endemic nematode isolates with
with a silken thread onto the soil, in which they bury themselves
good efficacy against a specific target insect, thereby circumventa few millimetres down to spin tightly woven cocoons, in which
ing the importation of exotic nematodes. While many surveys
soil is incorporated. After a period of approximately 23 days,
have recently been conducted in other parts of the world, in conthe pre-pupa in the cocoon changes into a pupa, and the adult
trast only a few surveys have been conducted in South Africa
moth emerges after a further 1216 days at 25C, with longer in(Malan et al., 2006, 2008; Hatting et al., 2009) and throughout
tervals at lower temperatures (Daiber, 1979a, 1979b, 1980, 1989).
the rest of the African continent, which remains relatively unexThe soil stages of FCM targeted with entomopathogenic nemaplored, offering a fertile field for bioprospecting.
todes (EPNs) are shown in Figure 1 above.
The primary objective of the study was to obtain EPNs spe-

64

SA FRUIT JOURNAL
DEC 2011/JAN 2012

FRUITGROSCIENCE

cifically from South African citrus orchards and to contribute to the

existing knowledge of the geographical distribution and diversity of
EPNs. As no previous research has been undertaken into the control
of FCM using nematodes, the second objective of the study was to determine the potential of EPNs for the control of the soil stages of FCM.
Nematode species found during the survey were tested for infectivity
by evaluating the percentage mortality in laboratory bioassays against
last-instar larvae, pupae and emerging moths.

Survey of citrus orchards A total of 129 samples came from the West-

ern Cape, with 20 (15.5%) samples testing positive for EPNs; 52 samples
from the Eastern Cape, with eight (15.4%) testing positive; and 21 from
Mpumalanga, with seven (33.3%) testing positive. Of the 202 samples
Fig. 2. Occurrence and distribution of EPNs in citrus orchards
taken, 35 (17%) tested positive for the presence of EPN (see Fig. 2).
in the Western Cape, Eastern Cape and Mpumalanga provinces
During the survey of citrus orchards, three species of Steinernema
of South Africa. Key: o = H. bacteriophora; x = H. zealandica;
and three of Heterorhabditis were identified and characterised, using a
= Heterorhabditis sp.; + = S. khoisanae;
combination of morphological and molecular techniques. The stein* = S. yirgalemense; = Steinernemacitrae.
ernematids, S. citrae (Stokwe et al., 2011), S. khoisanae and S. yirgalemense, and heterorhabditids, H. bacteriophora,
H. zealandica and an undescribed Heterorhabditis spp., were reported.
Steinernema khoisanae, an endemic species
for South Africa, was previously recovered
from grass, apple and grapevine soils in the
Western Cape province (Nguyen et al., 2006).
In this study, S. khoisanae was identified from
a citrus orchard in the Porterville area.
Steinernema yirgalemense was found in the
Nelspruit area of Mpumalanga province. This
is a new record for a Steinernema, as the only
other species reported to be present in South
Fig. 3. Mean percentage mortality of FCM last-instar larvae inoculated with 50 IJs/insect
Africa have, in the past, been S. khoisanae and
after 48 h at 25C.
S. citriae. Steinernema yirgalemense was described by Nguyen et al. (2004) from Yiglemen in Ethiopia, where it
was found to be the dominant species. It was also reported from the
Central Rift Valley Region of Kenya (Mwaniki et al., 2008).
Heterorhabditis zealandica has only once previously been reported in
connection with natural vegetation in the Eastern Cape. In the current
survey, the nematode was found in two orchards in the Western Cape
and in one in Mpumalanga. However, in an extensive survey of seven
geographical regions throughout South Africa, which was undertaken
by Hatting et al. (2009), no H. zealandica was found, indicating the species to be rare, with a limited distribution in South Africa. The new Heterorhabditis found during this survey is currently in the process of being
described as a new species. However, H bacteriophora seems to be the
most common species occurring in citrus orchards.

Testing of last-instar FCM larvae for susceptibility against

nematodes Last-instar FCM larvae were found to be highly sus-

ceptible to low concentrations (50 larvae per insect) of the six nematode species used, with mortality ranging between 77100% (Fig. 3).
Steinernema yirgalemense gave 100% control, followed by H. zealandica, which was the same South African isolate as that which has been
used against codling moth. An FCM larva infected with nematodes is
shown in Figure 4.

Fig. 4. Dissected larva infected with nematodes.

Testing pupae for susceptibility against nematodes

The pupae of FCM were found not to be as susceptible as

were the larvae and needed three times the concentration of
nematodes to give control of between 2075% (see Fig. 5).
For the pupae, the same pattern as for the larvae was found
SA VRUGTEJOERNAAL
DES 2011/JAN 2012

65

FRUITGROSCIENCE

chamber at 25C. This provided time for the

larvae to burrow into the sand, to spin into cocoons and to turn from pre-pupae into pupae.
The nematodes S. yirgalemense, S. khoisanae
and H.zealandica sp. were added by spraying
800 IJs/ml (20 IJs/cm2) onto the sand. After 14
days, all live and dead moths were recorded,
and the remaining pupae were retrieved by
H. khoisanae
washing the sand through a sieve. In the nemFig. 5. Mean % mortality of FCM pupae inoculated with 200 IJs/insect after 48 h at 25 C.
atode treatments, all live moths were caught
and left in a petri dish with moistened filterpaper. The moths were kept until they died
and were then dissected for infection. A dissected FCM pupa and an
FCM adult infected with nematodes are shown in Figure 7.
The pupae found killed in the sand were analysed separately. It was
found that S. yirgalemense was able to infect about 40% of the pupae, in
comparison with the 75% infected in the 24-well bioassays, before they
emerged as moths, with no significant difference being found in the performance of H. zealandica (Fig. 8). However, S.yirgalemense was found
capable of infecting a higher number of moths than was H. zealandica.
In the sand bioassays, S. khoisanae, with a large IJ (> 1 000 m) length,
was considered to perhaps have an advantage. However, in overall
performance, S. yirgalemense killed the highest percentage of pupae
and moths in the sand bioassay, followed by H. zealandica, and then
S. khoisanae (Fig. 9). The percentage of fully emerged moths that were
infected with nematodes was 51% for S. yirgalemense, 47% for H. zeaFig. 6. FCM pupa infected with EPNs.
landica, and 49%
for S. khoisanae.

Fig. 7. On the left, a dissected FCM pupa and, on the right, a dissected FCM adult infected with nematodes.
with the six EPN species, with S. yirgalemense and H. zealandica performing the best and S. khoisanae the worst. A pupa
infected with nematodes is shown in Figure 6.

Testing the susceptibility of cocooned pupae and

emerging moths in sand The two best performing spe-

cies, S. yirgalemense and H. zealandica, and the worst performing, S. khoisanae, were chosen to determine the performance
of the nematodes against pupae naturally burrowed into
sand and spun into cocoons. Twenty last-instar FCM larvae
were placed on top of sand in each container. The closed
containers were left for a period of seven days in a growth

66

SA FRUIT JOURNAL
DEC 2011/JAN 2012

Discussion Nematodes have not previously been used for the bio-

logical control of FCM, with the above being the first result of their
possible use as biocontrol agents against a key pest of citrus in South
Africa. Both S. yirgalemense and H. zealandica have shown promise as
biological control agents in laboratory bioassays against FCM larvae,
pupae and emerging moths. In addition, H. zealandica has also been
found to be highly virulent against codling moth (Cydia pomonella, L.)
diapausing larvae residing beneath the bark of apple trees when applied as an aerial application (De Waal et al., 2010, 2011).
The biology of FCM offers a window of opportunity for the application of EPN to the soil. The last-instar larvae, the cocooned pupae and
the emerging moths are in the soil for a total period of approximately

FRUITGROSCIENCE

two weeks or longer, depending on the temperature, during which they can be targeted using EPNs.
Notable from the sand bioassay, is that moths infected
with nematodes were able to emerge normally and to fly
away from the sand surface, before being trapped in the
surrounding container. In natural conditions, the moths
would be able to fly away for at least 24 h before they
would be killed by septicaemia. Such a finding means that
EPN are able to distribute to other localities via aerial transport of infected emerging moths. If they fall onto the soil in
a moist environment, the life cycle of the nematode can be
completed.
The results of the current study showed the six nematode species found during the survey to be effective against
FCM soil stages in laboratory bioassays, with clear differences between the species. The same pattern for mortality
with the different species was reflected in all the host life
stages tested, with S. yirgalemense and H. zealandica causing
the highest level of mortality and S. khoisanae the lowest.
Nematode species were found to be highly virulent against
last-instar FCM larvae and emerging moths, while pupae
were not found to be that susceptible to nematode infection.
Since FCM is a key pest of citrus in South Africa, it would
be advisable to test the nematodes in field trials in an integrated pest management (IPM) system for the control of
FCM. The pest species concerned is also multivoltine, with
up to six generations per year, offering year-round availability of a host for EPNs, with the possibility of persistence
in citrus orchards.

Acknowledgement The authors would like to thank Cit-

rus Research International (CRI) and the Technology and

Human Resources for Industry Programme (THRIP) for
funding of the project; Sheila Storey from Nemlab; Wayne
Kirkman, John-Henry Daneel and Bruce Tate from CRI for
taking soil samples; and River Bioscience for supplying the
FCM larvae.

References
Daiber, C.C. 1979a. A study of the biology of the false codling moth [(Cry-

ophlebia leucotreta (Meyer.)]: The larva. Phytophylactica 141-144.

Daiber, C.C. 1979b. A study of the biology of the false codling moth [(Cryptophlebia leucotreta (Meyr.)]: The cocoon. Phytophylactica 11, 151-157.

Daiber, C.C. 1980. A study of the biology of the false codling moth Cryptophlebialeucotreta (Meyr.): The adult and generations during the year. Phytophylactica 12, 193.

Daiber, C.C. 1989. The false codling moth, Cryptophlebia leucotreta (Meyr)

(Lepidoptera, Tortricidae), in Southern Africa. J. Pl. Dis. Protec. 96, 71-80.

De Waal, J.Y., Malan, A.P., Addison, A.P. 2011. Evaluating mulches together

with Heterorhabditis zealandica (Rhabditida: Heterorhabditidae) for the con-

trol of diapausing codling moth larvae, Cydia pomonella (L.) (Lepidoptera:

Tortricidae). Biocontr. Sci. Technol. 21, 255-270.

De Waal, J.Y., Malan, A.P., Levings, J., Addison, M.F. 2010. Key elements

in the successful control of diapausing codling moth, Cydiapomonella (L.)

Fig. 8. Mean mortality of cocooned FCM pupae in sand inoculated with

20 IJs/cm2, seven days after treatment.

Fig. 9. Mean percentage mortality of cocooned FCM pupae and emerging

moths in sand inoculated with 20 IJs/cm2,seven days after treatment.

Hatting, J., Stock, P.S., Hazir, S. 2009. Diversity and distribution of entomopathogenic

nematodes (Steinernematidae, Heterorhabditidae) in South Africa. J. Invertebr. Pathol. 102,

120-128.

Hofmeyer, J.H., Carpenter, J.E., Bloem, J.E. 2005. Developing the sterile insect technique

for Cryptophlebia leucotreta (Lepidoptera: Tortricidae): Influence of radiation dose and re-

lease ratio on fruit damage and population growth in field cages. J. Econ. Entomol. 98,
1924-1929.

Malan, A.P., Nguyen, K.B., Addison, M.F. 2006. Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the southwestern parts of South Africa. Afr.
Pl. Prot. 12, 65-69.

Malan, A.P., Nguyen, K.B., De Waal, J.Y., Tiedt, L. 2008. Heterorhabditis safricana n. sp.

(Rhabditida: Heterorhabditidae), a new entomopathogenic nematode from South Africa.

Nematology 10, 381-396.

Mwaniki, S.W., Nderitu, J.H., Olubayo, F., Kimenju, J.W., Nguyen, K., 2008. Factors

influencing the occurrence of entomopathogenic nematodes in the Central Rift Valley Region of Kenya. Afr. J. Ecol. (Suppl.1)46, 79-84.

Moore, S.D., Kirkman, W. 2008. Citrus orchard sanitation with emphasis on false codling
moth control. SAFJ 7, 57-60.

Newton, P.J. 1998. False codling moth Cryptophlebia leucotreta (Meyrick). In: Citrus pests

in the Republic of South Africa. E.C.G. Bedford, M.A. van den Berg & E.A. de Villiers. (eds.)
Dynamic Ad, Nelspruit, South Africa, pp. 192-200.

Nguyen, K.B., Malan, A.P., Gozel, U. 2006. Steinernema khoisanae n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Africa. Nematology 8, 157175.

Nguyen, K.B., Tesfamariam, M., Gozel, U., Gaugler, R., Adams, B.J. 2004. Steinernema
yirgalemense n. sp (Rhabditida : Steinernematidae) from Ethiopia. Nematology 6, 839-856.

(Lepidoptera: Tortricidae) in fruit bins with a South African isolate of Het-

Stokwe, N.F., Malan, A.P., Nguyen, K.B., Knoetze, R., Tiedt, L. 2011. Steinernema citrae

20, 489-502.

rica. Nematology 13, 569-587.

erorhabditis zealandica (Rhabditida: Heterorhabditidae). Biocontr. Sci. Technol.

n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Af-

SA VRUGTEJOERNAAL
DES 2011/JAN 2012

67