Fungal species associated
with three phytophagous
insects of gorse
Purpose
E. Yamoah , R.J. Weld , E.E. Jones ,
2
3
D.M. Suckling , N. Waipara ,
4
1
G. Bourdôt , A. Stewart
1
1
2
2
4
1
1
National Centre for Advanced Bio-Protection Technologies,
PO Box 84, Lincoln University, New Zealand (NZ),
HortResearch, PO Box 51, Lincoln, NZ,
Landcare Research Ltd, Private Bag 92170, Auckland, NZ,
AgResearch Ltd., PO Box 60, Lincoln, NZ.
Results
Fusarium tumidum is a potential mycoherbicide for gorse control.
Phytophagous insects naturally found on this weed, may serve
as deliberate vectors of F. tumidum spores for gorse biocontrol.
In this study, the diversity of fungi on the surfaces of three
phytophagous insect species of gorse was studied by culturing
and culture independent techniques.
• Acremonium, Alternaria, Aspergillus, Aureobasidium pullulans,
Beauveria, Cladosporium, Epicoccum purpurascens, Nectria,
Penicillium, Phoma, Pithomyces, Sclerotinia, Verticillium and yeast
species were isolated from all three insect spp. (Fig. 3).
• The yeasts include Metschnikowia pulcherrima, Pseudozyma
fusiformata, Rhodotorula mucilaginosa and Sporobolomyces
ruberrimus.
• Fusarium lateritium and Fusarium tricinctum were isolated from
the moths only and Gibberella pulicaris was isolated from E.
postvittana.
• E. postvittana carried the most (33) fungal species, similar to 29
species isolated from C. ulicetana, while A. ulicis carried the least
(16).
• The three collection sites had similar fungal diversity.
Methods
• Insect species Apion ulicis (weevil), Cydia ulicetana (moth) and
Epiphyas postvittana (moth) (Fig. 1) were collected from three sites
in the South Island of New Zealand.
• The largest RFLP group, 94% of the cloned amplicons, was made
up of 10 strains of Nectria mauritiicola.
• The direct PCR method produced fewer RFLP groups (17) than the
culturing method (55).
• Only two uncultured fungi were identified by direct PCR.
Apion ulicis
Cydia ulicetana
Epiphyas postvittana
Fig. 1. Potential insect vectors.
• Fungal diversity on insects was examined through washing and
plating techniques.
• PCR products of amplified DNA obtained from the washing of the
insects were cloned into pGEM-T easy (Promega) vector in Escherichia
coli strain INVaF’.
Verticillium sp
Beauveria bassiana
Phoma herabarum
Epicoccum purpurascens
Pseudozyma fusiformata
Allernaria sp
Aurobasidium pullulans
Cladosporium cladosporrioides
Gibberella pulicaris
Pithomyces chartarum
Fusarium lateritium
Aspergillus sp
• Plasmids were extracted from 141 clones (Wizard® Plus SV Minipreps
DNA purification kit).
• PCR products from both directly cultured isolates and cloned
amplicons were digested with restriction enzymes: Hin6I, MboI,
BsuRI and HinfI (Fermentas) (Fig. 2).
• RFLP groups were sequenced and compared with the GenBank
database using BLAST search programme.
M
F. lateritium
U
1
2 3
4
M
G. pulicaris
U
1
2 3
4
M
F. tricinctum
U 1
2 3
4
Fig. 3. Selected fungal species isolated from three insects.
Conclusions
The moths have greater potential to vector F. tumidum spores as they
naturally carry Fusarium spores. A pheromone is available for attracting
the male moths to F. tumidum inocula for “lure-load-infect”. The
culturing method was better than direct PCR for studying fungal
diversity.
Acknowledgement
Fig. 2. Restriction fragment patterns of PCR amplified rDNA, digested with 1: Hin6I, 2: MboI,
3: BsuRI and 4: HinfI and analysed in 2% agarose gel. Lane M: molecular weight marker (50
bp ladder) (Invitrogen); lane U: undigested rDNA.
The New Zealand Tertiary Education Commission funded
this research.