sugarcane white grubs (coleoptera: …€¦ · entomopathogens infect and kill their insect hosts,...

SHORT, NON-REFEREED PAPER

SUGARCANE WHITE GRUBS (COLEOPTERA: SCARABAEIDAE) IN

AFRICA AND INDIAN OCEAN ISLANDS: THEIR PEST STATUS AND

THE POTENTIAL FOR FUNGAL ENTOMOPATHOGENIC CONTROL

CONLONG DE1,2 AND GANESHAN S3

1South African Sugarcane Research Institute, P/Bag X02, Mount Edgecombe, 4300, South Africa 2School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, South Africa 3Mauritius Sugarcane Industry Research Institute, Mauritius Cane Industry Authority,

Reduit 80835, Mauritius

[email protected] [email protected]

Abstract

White grubs are predominant sugarcane soil pests. Several species are recorded from Africa

and the Indian Ocean islands. The most widespread is Heteronychus licas, reported from 15

countries, probably also present but unreported in more sugarcane growing countries. Damage

by H. licas in sugarcane in Mauritius caused 100% loss, and led to costly replant programmes.

Promising indigenous entomopathogenic strains of Metarhizium anisopliae were isolated from

H. licas in Mauritius and Zimbabwe. In Tanzania, Cochliothis melolonthoides caused 70% loss

in ratoon sugarcane, and in Somalia, another melolonthine, Brachylepis werneri La Croix, is a

pest. An effective fungal entomopathogen, Ophiocordyceps barnesii, has been isolated from

both species. In southern and eastern African countries Schizonycha affinis and Pegylis

(=Hypopholis) sommeri are sugarcane pests. Virulent strains of Beauveria brongniartii have

been isolated from them in South Africa. Hoplochelus marginalis Fairmaire (Melolonthinae),

exotic to, but a major pest in Reunion, is the only white grub species in the region controlled

with a commercial formulation of B. brongniartii (BETEL®). Rutelines such as Anomala spp.

and Adoretus spp. occur in several countries but do not reach major pest status. Biological and

ecological differences between melolonthines, rutelines and dynastines render their

management difficult. However, indigenous fungal entomopathogens offering novel control

options are discussed.

Keywords: biopesticides, biological control, SADC, biodiversity, integrated pest management, EU-

ACP

Introduction

Scarabaeids (Coleoptera) are the dominant group of soil pests associated with sugarcane

(Carnegie and Conlong, 1994). In Africa and the Indian Ocean islands, problem species occur

in Malawi (Rutherford and Way, 2003), Mauritius (Rajabalee, 1994; Behary-Paray et al.,

2012), Mozambique (Way and Goebel, 2003; Matusse 2013), Nigeria (Taylor, 1966), Reunion

Island (Anon, 2010), South Africa (McArthur and Leslie, 2004; Way et al., 2011; Goble et al.,

2012, 2015), Swaziland (Williams, 1985; Carnegie, 1988; Way et al., 2013), Tanzania (Evans

et al., 1999; Behary-Paray et al., 2012), Somalia (Cock and Allard, 2013), Uganda (Conlong

Conlong DE and Ganeshan S Proc S Afr Sug Technol Ass (2016) 89: 116-124

116

mailto:[email protected]

mailto:[email protected]

and Mugalula, 2003; Mugalula et al., 2006) and Zimbabwe (Cackett, 1980; Musikavanhu,

1996; Mabveni and Mgocheki, 2012).

Scarabaeid larval feeding on sugarcane roots causes it to become stunted, lodge easily, yield

poorly and ratoon poorly, a factor often neglected in damage assessments (Sosa, 1994). Adult

damage on young sugarcane is easily noticeable as it produces similar symptoms to stem borer

shoot attack (Taylor, 1966). The growing point of the shoot dies (‘dead heart’), caused by adult

gnawing of tillers just below soil level reaching and destroying the shoot meristem (Conlong,

2015).

Previously, infested sugarcane fields were treated with pesticides. Despite many formulations

tried, none lasted (Conlong and Mugalula, 2003). This was because of insect resistance to the

chemical, environmental side effects and cost implications. Morphological similarity and

difficulty in identifying larval stages of different species of scarabaeids, causing them to be

clumped together under the collective name of white grubs further complicated control

measures. No account has been taken of different scarabaeid species’ ecologies and biologies

(Conlong and Mugalula, 2003), which compromised insecticidal control options, as often they

were applied when susceptible insect stages were absent from application areas.

Entomopathogens infect and kill their insect hosts, and have potential for natural pest control.

From a biological control perspective these ‘hidden treasures’ causing disease within insect

populations have been overlooked, not because their potential of entomopathogenism is low,

but because of research paucity on them and their development as biological control agents.

Only two sugar producing countries have used commercial formulations of fungal

entomopathogens. In Australia, a Metarhizium anisopliae based biopesticide (BioCane),

caused epizootics in scarabaeid larval populations (Samson et al., 2006). On Reunion Island,

the commercial formulation of Beauveria brongniartii (BETEL) is a compulsory treatment

when sugarcane is replanted (Anon, 2010).

However, interest in the use of fungal entomopathogens in Africa and on Indian Ocean Islands

is evident, and M. anisopliae has been collected from scarabaeid species in Zimbabwe

(Mazodze and Zvoutete, 1999) and Mauritius (Rajabalee, 1994), as has Ophiocordyceps

barnesii from Cochliotis melolonthoides (Gerstaecker) (Melolonthinae) in Tanzania (Evans et

al., 1999). In South Africa, virulent strains of Beauveria brongniartii have been isolated from

the melolonthids Schizonycha affinis Boheman and Pegylis (=Hypopholis) sommeri

(Burmeister) (Goble et al., 2012, 2015). To build on and complement these studies, an

European Union-African, Caribbean and Pacific Group of States (EU-ACP) funded project ran

from 2010 to 2015, investigating the potential for development of indigenous biopesticides for

control of white grubs in sugarcane producing Southern African Development Community

(SADC) countries. Its aims were to establish identities of white grub species causing damage

to sugarcane in these countries, and to identify fungal entomopathogens attacking these which

could be developed into commercially produced biopesticides for local production and use.

This paper summarises the results of this project.

Materials and Methods

Training

In February 2011 training was given at the South African Agricultural Research Centre-Small

Grains Institute (ARC-SGI) in Bethlehem, Free State province. Nineteen persons from each


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collaborating institution in seven countries attended. A manual (Hatting, 2011) was distributed.

Attendees were asked to complete surveys in their respective sugarcane for diseased insects, to

be shipped to the ARC-SGI where pathogens were isolated and bioassayed, before being passed

on to South African National Collection of Fungi (SANCF) for identification, accessioning and

preservation. Collaborators were shown how to collect entomopathogens from soil using ‘trap

insects’ (Galleria mellonella (Lepidoptera: Pyralidae) larvae). In addition, they sent live white

grub specimens to the South African Sugarcane Research Institute (SASRI) quarantine

laboratory, who passed on quarantined material to Stellenbosch University for morphological

and molecular identifications.

Country visits by project leaders to Madagascar, Zimbabwe, Malawi, South Africa, Swaziland,

Mauritius, Mozambique, and Tanzania to further train field personnel in scarabaeid problem

identification, insect collection and dispatch for disease isolation and taxonomic studies

followed.

Identification of entomopathogens

Isolates received, collected and bioassayed by ARC-SGI were passed on to SANCF, who

compiled an international database of indigenous entomopathogenic fungi received. Pure

isolates with dry conidia were harvested from these and stored in sterile tubes at 4°C. These

have been correctly identified, given unique Plant Protection Research Institute (PPRI)

accession numbers and preserved by SANCF. Bacteria received from collaborating countries

were isolated and cryo-preserved through inoculation in 15% glycerol/nutrient broth and placed

at -80°C.

Molecular and morphological identification of scarabaeids

Three thousand collected specimens are housed at Stellenbosch University, Department of

Conservation Ecology and Entomology (ConsEnt). Half of all specimens are adults, and over

half of all specimens are stored in absolute ethanol. Tubes containing ethanol specimens are

stored at -14°C for preservation of genetic material. Adults were pinned, curated, identified to

species level and stored in museum cabinets.

Analyses focused on DNA sequencing of adults to correlate with conspecific white grubs,

taxonomic key construction and data analysis. This work was complemented by simultaneously

focusing on collection and preservation of fresh material, photographing key morphological

characters, development of a white grub image library and construction of a white grub

identification key.

Results and Discussion

Collection of entomopathogens

Two thousand, one hundred and eighty-three cadavers were processed by ARC-SGI, yielding

60 Beauveria, 132 Metarhizium, 173 Trichoderma and 622 bacterial isolates (Table 1).


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Table 1. Overall numbers of material processed and entomopathogens isolated by ARC-SGI,

and passed on to SANCF for identification, accessioning and storage.

Country No. of

cadavers

Beauveria

group

Metarhizium

group

Trichoderma

spp. Bacteria

South Africa 1040 37

(8G+1UN+28WG) 37 (11G + 26WG) 107 225

Tanzania 436 21 (4G + 17 WG) 35 (8G + 27WG) 34 170

Swaziland 58 0 1(1WG) 5

Madagascar 2

Mauritius 242 2 (2G) 49 (25G + 2UN +

22 WG) 24 76

Malawi 75 0 1 (1 WG) 57

Mozambique 69 0 4 (4WG) 3 13

Zimbabwe 263 0 5 (5WG) 3 76

Total 2183 60 132 173 622

WG = Scarab cadavers; G = Galleria trapping method; UN = Unknown

Acremonium sp., Aspergillus sp. and Clonostachys sp. were identified by SANCF from

‘unknown’ pathogens sent from ARC-SGI. A benefit was isolation of several strains of

Trichoderma spp. (free-living fungi common in soil and root systems), recognised as biological

control agents of plant pathogens, for their ability to promote crop root growth and

development, and increasing plant resistance to abiotic stresses.

Molecular and morphological identification of scarabaeids

Seven hundred and fifty specimens of white grubs and adults were sequenced, producing a total

of 2019 gene sequences (603 for 28S, 586 for 16S, 830 for COI). Using morphological and

molecular characters, 59 species of white grubs were identified (Table 2). Of these, the majority

(41) could not reliably be associated with an existing species. All but one (Heteronychus licas

(Klug) Dynastinae) were medium to low-level pests, normally with different species occurring

in different countries. Only H. licas occurred in all countries (Table 2).


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Table 2. Scarabaeid species found during the study, their pest status, type of data collected and country of occurrence (Species in blue and red font

have high pests status in individual countries, and broad distribution respectively in SADC sugarcane growing countries).

Pest status†

Type of data

collected†† Country of occurrence†††

Adults Grubs CO1 28S 16S SA MAL MAU ZIM MOZ MAD TAN SWA

DYNASTINAE

Oryctini Oryctes rhinoceros L L 1 1 1 1

Oryctes cf. boas L L 1 1 1 1

Pentodontini Alissonotum piceum H H 1 1 1 1

Heteronychus arator M 1 1 1 1

Heteronychus licas H H 1 1 1 1 1 1 1 1 1 1 1

Heteronychus sp.? L 1 1 1 1

Temnorhynchus clypeatus M 1 1 1 1 1

RUTELINAE

Anomalini Anomala A sp.3 M M 1 1 1 1

Anomala A sp.4 H 1 1 1 1

Anomala A sp.5? L 1 1 1 1

Anomala A sp.7? M M 1 1 1 1

Anomala B sp.J1 M 1 1 1 1

Anomala B sp.J3 L M 1 1 1 1 1 1

Anomala B sp.8? M M 1 1 1 1

Anomala B sp.10? M M 1 1 1 1

Adoretini Adoretus cf. ictericus M 1 1 1 1

Adoretus nasutus M 1 1 1 1

Adoretus sp.1? L 1 1 1

Adoretus sp.2? L 1 1 1 1

Adoretus B cf. fusculus M 1 1 1 1

Adoretus B cf. fusculus2 M 1 1 1 1

Adoretus B fusculus M 1 1 1 1 1

MELOLONTHINAE

Unknown Melolonthinae sp.1 M 1 1 1 1

Melolonthinae sp.2 L 1 1 1 1

Melolonthinae sp.3 L 1 1 1 1

Melolonthini Schizonycha gracilis H 1 1 1 1

Schizonycha lebidis M 1 1 1 1

Schizonycha divulsa L 1 1 1


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Schizonycha neglecta M 1 1 1 1

Schizonycha cf. abrupta L 1 1 1 1

Schizonycha sp.9? L 1 1 1 1


Schizonycha sp.1 M 1 1 1 1 1

Schizonycha cf. affinis H 1 1 1 1

Schizonycha livada L 1 1 1 1



Clemora smithi L H 1 1 1 1

Hypopholis sommeri L H 1 1 1 1

Hypopholis vittata M 1 1 1 1

Asthenopholis subfasciata M 1 1 1 1 1

Cochliotis melolonthoides L H 1 1 1 1

Sericini Trochalus sp.1? L 1 1 1 1

Trochalus sp.2? L 1 1 1 1

Trochalus sp.3? L 1 1 1 1

Trochalus sp.SA2? L 1 1 1 1

Autoserica sp.1 L 1 1 1 1

Autoserica tessellata M 1 1 1 1

Maladera sp.1? L 1 1 1 1

Maladera sp.2? L 1 1 1

Maladera sp.3? L 1 1 1

Maladera sp.4? L 1 1 1 1

Ablaberini Ablaberini sp.1 M 1 1 1 1

Ablaberini sp.2 M 1 1 1 1

Ablaberini sp.3 L 1 1 1 1

Hopliini Congella cf. valida M 1 1 1 1

Congella 'tesselatula' M 1 1 1 1

Tanyproctini "nr. Clitopa" M 1 1 1 1

† Pest status: Letters indicate degree of economic significance of pest. H = high, M = medium, L = low significance as economic pest.

†† Type of data collected: CO1, 16S and 28S are DNA gene regions which were sequenced, and morphology indicates morphological trait scoring dataset collected. Black cells

indicate successful data collection.

††† Country of occurrence: SA = South Africa; MAL = Malawi; MAU = Mauritius; ZIM = Zimbabwe; MOZ = Mozambique; TAN = Tanzania; SWA = Swaziland.


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Conclusions

White grub species diversity in collaborating countries was greater than expected. Field visits

indicated correctly identifying damage associated with white grubs was problematic. Training

during country visits highlighted this, and growers are now completing regular white grub

surveys.

In addition to Table 2, five more keys were constructed to identify species of white grubs,

intended for different users with varying degrees of scientific rigour:

(i) A hierarchical, pictorial key based predominantly on raster pattern characters for the white

grub pests of South Africa.

(ii) Individual country-specific pictorial keys for each participating country, using mostly

images with few words. In countries like Tanzania and Mauritius, with relatively few white

grub pest species, this key is designed as a poster to hang on a wall.

(iii) A large field key designed as an A4-sized handbook, to all species collected, arranged

taxonomically using simple to view, external morphological characters only.

(iv) A large scientifically rigorous key to all species, including a dichotomous taxonomic key

and utilising all characters, internal and external.

(v) An interactive, digital, multi-entry key including all of the species and morphological

characters designed in the software program LUCID, and posted online.

A wide range of entomopathogens with varying virulence were discovered, demonstrating the

potential of developing indigenous bioinsecticides. In South Africa two B. brongniartii strains

are formulated and are being used in ongoing trials against targeted white grubs. In Mauritius

isolates of M. anisopliae from Mauritius, Tanzania and Zimbabwe are being mass produced

for trials.

Several strains of Trichoderma spp. have been isolated from samples received from

collaborators. Trichoderma are known as beneficial fungi enhancing soil heath and plant

growth. Many strains are available commercially. Strains found during the current project

could have similar beneficial effects and have potential for commercial development.

Acknowledgements

Results would not have been possible without expert entomopathogen input from Dr Justin

Hatting’s (ARC-SGI) team - isolating etiological agents and testing Koch’s postulates; Dr Elna

van der Linde’s (SANCF) team - identifying and preserving entomopathogenic isolates

discovered; Dr Mike Morris of Plant Health Products, Nottingham Road, and Nalini Behary-

Paray (MSIRI) - initial selected isolate formulation and mass production; Drs C Bazelet, P

Addison (ConsEnt, SU), and Dr Deborah Sweby and Mike Way (SASRI) - morphological and

molecular identification and key creation of white grub species found; and all country

collaborators contributing specimens to achieve the project aims.


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