Canadian Journal of Plant Pathology

ISSN: 0706-0661 (Print) 1715-2992 (Online) Journal homepage: http://www.tandfonline.com/loi/tcjp20

The banana weevil, Cosmopolites sordidus

(Germar), is a potential vector of Xanthomonas
campestris pv. musacearum in bananas
Evans Were, Gloria V. Nakato, Walter Ocimati, Idd Ramathani, Samuel Olal &
Fenton Beed
To cite this article: Evans Were, Gloria V. Nakato, Walter Ocimati, Idd Ramathani, Samuel
Olal & Fenton Beed (2015) The banana weevil, Cosmopolites sordidus (Germar), is a potential
vector of Xanthomonas campestris pv. musacearum in bananas, Canadian Journal of Plant
Pathology, 37:4, 427-434, DOI: 10.1080/07060661.2015.1113444
To link to this article: http://dx.doi.org/10.1080/07060661.2015.1113444

Accepted author version posted online: 13

Nov 2015.
Published online: 13 Nov 2015.
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Date: 15 February 2016, At: 10:56

Can. J. Plant Pathol., 2015

Vol. 37, No. 4, 427434, http://dx.doi.org/10.1080/07060661.2015.1113444

Bacteria and phytoplasmas / Bactries et phytoplasmes

The banana weevil, Cosmopolites sordidus (Germar), is a potential

vector of Xanthomonas campestris pv. musacearum in bananas

EVANS WERE1,2, GLORIA V. NAKATO1, WALTER OCIMATI3, IDD RAMATHANI1, SAMUEL OLAL2
AND FENTON BEED1
1

Plant Pathology, International Institute of Tropical Agriculture (IITA), P.O. Box 7878, Kampala, Uganda
Department of Biological Sciences, Kyambogo University, P.O. Box 1, Kyambogo, Uganda
3
Commodity Systems and Genetic Resources Programme, Bioversity International, P.O. Box 24384, Kampala, Uganda

Downloaded by [Evans Were] at 10:56 15 February 2016

(Accepted 24 October 2015)

Abstract: This study was carried out to investigate the potential role of banana weevils as vectors of Xanthomonas campestris pv.
musacearum (Xcm), causal agent of banana wilt. Weevils captured from Xcm-infected plants were tested for presence of Xcm, and further
raised on Xcm-infected corms for later use as vectors to transmit the pathogen to healthy tissue-cultured plantlets. Analysis of weevils
captured from diseased elds revealed more weevils contained Xcm originating from Mbwazirume compared with Kayinja cultivars.
Colonies of Xcm were recovered from the weevil external body surface, internal organs (mouth parts and abdomen) and faecal matter.
There was signicantly higher Xcm presence and cfu mL1 on the external weevil body surface than within the internal organs. Bacterial
populations declined progressively from the external body surface, internal mouth parts, internal abdominal parts and the faecal matter.
Following placement of weevils previously fed on Xcm-exuding corms in close proximity to healthy potted plants, infection occurred,
with characteristic disease symptoms observed on all cultivars evaluated except Kayinja which remained symptomless. Isolation from
both symptomatic and asymptomatic plants revealed erratic Xcm incidence and cfu g1 that did not correlate to the number of weevils
released in all cultivars, except for Kayinja. This study showed that Xcm can survive on and within the banana weevil and potentially
spread the pathogen to neighbouring plants.
Keywords: beetle vector, cultivar response, disease management, pathogen transmission, Xanthomonas wilt
Rsum: Cette tude a t mene dans le but dvaluer le possible rle que jouent les charanons du bananier en tant que vecteurs de
Xanthomonas campestris pv. musacearum (Xcm), lagent causal du trissement du bananier. Les charanons capturs sur des plants
infects par Xcm ont t tests pour ly dceler, puis levs sur des cormes infects an de les utiliser comme vecteurs dans le but de
transmettre lagent pathogne des plantules saines issues de la culture tissulaire. Lanalyse des charanons capturs dans des champs
infects a rvl quun plus grand nombre dinsectes contenaient le Xcm provenant du cultivar Mbwazirume que du cultivar
Kayinja. Des colonies de Xcm ont t rcupres de la surface du corps, des organes internes (pices buccales et abdomen) et des
fces des charanons. La surface de la carapace des charanons afchait un taux signicativement plus lev de Xcm et de cfu ml1
que leurs organes internes. Les populations bactriennes dcroissaient graduellement en passant de la surface du corps aux pices
buccales internes, aux organes internes de labdomen puis aux fces. Aprs avoir plac les charanons stant pralablement nourris de
cormes exsudant le Xcm sur des plants en pot sains, linfection est survenue, tous les cultivars valus afchant les symptmes
caractristiques de la maladie, sauf Kayinja qui est demeur intouch. Lisolement des plants symptomatiques et asymptomatiques a
rvl une incidence irrgulire de Xcm ainsi que des cfu g1 qui ntaient pas corrls au nombre de charanons relchs sur tous les
cultivars, sauf pour Kayinja. Cette tude a montr que Xcm peut survivre sur et dans les charanons du bananier et transmettre lagent
pathogne aux plants croissant proximit.

Correspondence to: Gloria Valentine Nakato. E-mail: V.Nakato@cgiar.org

Present address for Fenton Beed: AVRDC The World Vegetable Centre, P.O. Box 1010 (Kasetsart), Bangkok 10903, Thailand
2015 The Canadian Phytopathological Society

E. Were et al.

428

Mots cls: coloptre vecteur, trissure Xanthomonas, gestion des maladies, raction des cultivars, transmission de lagent pathogne

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Introduction
Bananas (including plantains) are cultivated annually on
10.5 million hectares in the tropical and subtropical
regions of the world, with annual world production estimated at 27 million tonnes (FAO 2013). It is mainly
consumed as a dessert banana, but in some parts of
Africa, particularly in eastern and southern Africa,
banana is also used to make beer or is steamed and
eaten. Steamed banana is usually made from the East
African highland bananas (genotype AAA-EA) and plantain (genotype ABB) which are grouped under the cooking type of bananas. The East African highland banana
comprises about 70% of the bananas produced in the
East African region and is a staple food for several of
the East African countries, especially Uganda (Sharrock
& Frison 1999).
Despite its importance in the diet, banana production has
declined in Uganda to record low yields of around
530 t ha1 y1 compared with a potential yield of
70 t ha1 y1(Van Asten et al. 2004). The major biotic factors
responsible for this decline are pests and diseases, including
Xanthomonas wilt (BXW) caused by Xanthomonas campestris pv. musacearum) (Xcm) (Tushemereirwe et al. 2006)
and the banana weevil (Cosmopolites sordidus Germar)
(Gold et al. 2001). These two pests have been studied
extensively in Uganda and found to cause signicant losses
to the banana production industry.
Symptoms due to BXW include progressive yellowing
and wilting of leaves, shrivelling and rotting of the male
buds, premature ripening and internal discolouration of
fruits, and pockets of pale yellow bacterial ooze produced 515 min after diseased stems are cut
(Tushemereirwe et al. 2004). On the other hand, banana
weevils cause damage by larvae feeding within the corm
and pseudostem, thus causing signicant yield reduction,
reduced suckering and shortened plantation life (Gold &
Messiaen 2000; Gold et al. 2002; Messiaen 2002). It is
known that adult weevils are free-living but are often
found at the base of banana mats or associated with crop
residues. They are nocturnally active, relatively sedentary and rarely y. The weevils move freely within
banana stands and can migrate into neighbouring elds,
although few will disperse more than 50 m in 3 months
(Gold et al. 1999). Adult weevils may survive up to
4 years while feeding on rotting banana tissue
(Budenberg et al. 1993) and sometimes may feed on
young suckers (Castrillon 2000), although the damage
is negligible. Adult female weevils deposit their eggs

inside the plant tissue at the base of the pseudostem or

an exposed corm.
Despite the fact that weevils do feed on and oviposit in
the banana plant and are able to move within and between
farms, their potential as vectors for Xcm or any other
pathogen is unknown. Potentially, the feeding, reproduction and movement of banana weevils within and across
farms from adjacent areas could potentially enhance the
transmission of Xcm within and between mats and elds.
This study was aimed at establishing the potential of adult
weevils as vectors for Xcm by investigating: (i) weevil
banana cultivar preference; (ii) their potential to carry Xcm
in their gut or on their body surface; and (iii) their potential
to transmit BXW from infected corms to healthy banana
plants.
Materials and methods
This study was conducted on site and in the laboratory
located at Kifu National Forest Reserve in Mukono district
and the National Agricultural Research Laboratories
Institute (NARL), Kawanda, respectively. Four banana
cultivars Kayinja (genotype ABB), Mpologoma
(East African highland banana (EA), genotype AAA),
Nakitembe (genotype AAA-EA) and Musakala (genotype AAA-EA) obtained from Agro-Genetic
Technologies in Buloba, Mityana district, Central Uganda
were planted in pots in the Kifu forest reserve for the onsite trials. A fresh Xcm isolate obtained from a symptomatic plant in Mukono, central Uganda was used for this
study. All Xcm-like colonies were identied by PCR using
Xcm 38 primers (Adikini et al. 2011).
Weevil trap, capture and maintenance
Adult male and female banana weevils were captured
from Kayinja and Mbwazirume banana plantations
showing BXW symptomatic plants using split pseudostem traps (2530 cm) from recently harvested pseudostems (3070 cm above the collar) obtained from a
disease-free zone. Traps were placed next to banana
mats in elds with high weevil infestation and weevils
were recovered using sterile forceps and placed into
sterile 5 mL vials at 3-day intervals for a period of 31
days. Subsamples of these weevils were kept at 4C in
the laboratory at NARL for later isolation of Xcm. A
total of 1500 weevils were maintained for a period of
4 months on fresh Mbwazirume (a weevil-susceptible
cultivar) banana corms (i.e. corms were changed weekly)

Transmission of Xanthomonas by the banana weevil

with visible Xcm ooze inside a 20 L perforated closed
bucket for later inoculation studies.

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DNA extraction and PCR

Genomic DNA was extracted from all Xcm colonies isolated from the captured weevils, weevil body parts and
treatment plants using TES buffer (Mahuku 2004). A loopful of each Xcm colony was suspended in 500 L of 1 M
NaCl in Eppendorf tubes and vigorously vortexed. The
supernatant was discarded and the process repeated twice,
to reduce and separate the cells and nally washed twice in
sterile distilled water to reduce the salt concentration. The
bacterial pellets were suspended in 500 L of TES extraction buffer (0.2 M Tris-HCl (pH 8), 10 mM EDTA (pH 8),
0.5 M NaCl, 1% SDS containing 50 g mL1 proteinase K,
and vortexed for 30 s to thoroughly mix. Tubes were
incubated in a water bath at 65C for 15 min, after which
250 L of 7.5 M ammonium acetate was added and tubes
gently inverted thrice to mix. Tubes were incubated on ice
for 10 min and later centrifuged at 15 000 rpm for 15 min
and the supernatant transferred to a fresh tube. An equal
volume (500 L) of ice-cold isopropanol was added and
tubes incubated in a refrigerator at 20C overnight. Tubes
were centrifuged at 15 000 rpm and 4C for 10 min to pellet
the DNA and the supernatant discarded. The DNA pellet
was washed with 800 L of cold 70% ethanol and tubes
inverted on clean paper towels for 30 min to air-dry the
DNA pellet.
DNA integrity (concentration and purity) of each sample was determined using the NanoDrop 2000C spectrophotometer (Thermo Fisher Scientic Inc., Pittsburgh,
PA) and adjusted to 50 ng L1, for PCR using Xcm 38
PCR primers (Adikini et al. 2011). Amplication was
carried out in a 20 L reaction with a nal concentration
of 0.3 M of each of the primers, Xcm 38 F and Xcm
38 R, 1.5 mM MgCl2, 0.2 M of each dNTPs (Promega,
Madison, WI), 1 PCR green buffer, 1 unit of
HotStarTaq Plus DNA polymerase (Qiagen, Canada)
and 2 L of template. The PCR amplication was performed in the Ependorf Mastercycler (Ependorf AG,
Hamburg, Germany) and consisted of initial denaturation
at 95C for 5 min; 35 cycles consisting of 94C for 20 s,
annealing at 60C for 20 s, extension at 72C for 1 min
and a nal extension at 72C for 10 min. The amplied
PCR products were separated by gel electrophoresis
using 1.5% agarose in 1 TAE. The gel was stained
with ethidium bromide (0.5 g mL1) and visualized
with a UV transilluminator.

429
Isolation and detection of Xcm from weevils directly
trapped from diseased banana elds
A total of 68 weevil samples per banana cultivar (i.e.
Kayinja or Mbwazirume), each comprised of a vial of
3 weevils maintained at 4C, were used for this study.
Three mL of PBS-T, pH 7.4 (Na2CO3, 1.19 g; KHPO4,
0.2 g; KCl, 0.2 g; NaCl, 8.0 g; Tween-20 0.5mL) were
added to each vial and vortexed for one min to dislodge
any Xcm bacteria from the weevil body surface. One mL
of this surface rinse was serially diluted twice. For each
dilution, 20 L of the suspension was spread onto three
Petri dishes containing a semi-selective growth medium
of yeast peptone glucose agar (YPGA yeast extract 5 g,
peptone 5 g, glucose 10 g, and agar 15 g, 5-uorouracil
and cephalexin in 1000 mL of water) (Mwangi et al.
2007) and incubated at 28C for 72 h. Each sample was
monitored for growth of Xcm characteristic colonies and
a score of 1' assigned for Xcm presence and '0' for
colony absence.
To determine if the same weevils had Xcm in their
internal organs, weevils were surface sterilized by washing thrice with 3 mL of 15% sodium hypochlorite, followed by a single wash with 70% ethanol, and nally
rinsed thrice with 3 mL of sterile distilled water by
vortexing for 30 s. Twenty L of the nal rinse of each
weevil sample was plated to conrm that no Xcm from
the external body surface was present. The weevils were
ground using a sterile mortar and pestle and 3 mL of
sterile distilled water added. Xcm was isolated from the
diluent as described for the external body parts above.
All data were subjected to analysis of variance using
GenStat statistical software (Edition 12) and where signicance was noted, LSD (Least Signicance
Difference) at 5% was employed to separate the means.
Xcm isolation from faecal matter, gut and external body
parts of weevils maintained on diseased corms
Xcm isolation from the body surface. A total of 50
weevils maintained on the Xcm oozing corm samples
were placed in separate vials. Three mL of PBS-T were
added to each vial and vortexed for one min to dislodge
any Xcm from the weevil body surface. One mL of this
surface rinse was serially diluted twice. For each dilution, 20 L was plated and incubated as described above.
The plates were monitored for growth of Xcm colonies
and number of colonies counted. The mean cfu mL1 for
each weevil sample was computed as follows:
Cfu of bacteria per mL

Number of cfu  dilution factor

Volume plated in mL

E. Were et al.

430

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Xcm isolation from the internal gut section. Fifty weevils

were surface-sterilized as described above and rinsed
thrice with 3 mL of sterile distilled water by vortexing
for 30 s. 20 L of the nal rinse of each weevil sample
was plated to conrm that no Xcm from the external
body surface was present. A stereomicroscope was used
to dissect each weevil into head, thorax and abdomen
regions. Internal viscera were removed from the body
segments using sterile forceps and scalpels under the
stereomicroscope. The organs were ground using a mortar and pestle in 3 mL of PBS-T buffer and 1 mL of the
macerate serially diluted, plated on semi-selective
YPGA and incubated as above. Xcm presence and colony counts per gut section/body part were determined.
The cfu mL1 of each sample and body part were
computed.

Xcm isolation from the faecal matter. Fifty weevils maintained on Xcm oozing corms were surface-sterilized as
described above, rinsed thrice with 50 mL of sterile
distilled water and blotted dry with sterile paper towels
in the laminar ow hood. 20 L of the nal rinse of each
weevil sample was plated to conrm that no Xcm from
the external body surface was present. Five weevils were
sealed in a clean Petri dish with perforated paralm to
allow air circulation and kept in the dark at room temperature overnight to collect the faecal matter. The wee-

cfu of bacteria per gram

required. No fertilizers, pesticides or herbicides were

applied to the plants. Treatments were applied in orders
of 10 weevils (T10), 5 weevils (T5) and 1 weevil (T1) per
pot by placing the weevils maintained on Xcm oozing
corms close to the plant. Controls included plantlets that
received no weevils as a negative control and plantlets
inoculated in the corm by injecting with 1 mL of freshly
prepared Xcm suspended in sterile distilled water and
adjusted to 0.1 O.D600 (corresponding to 1.0108 cfu
mL1) as a positive control. Plants were maintained in a
conned area outdoors and observed for disease symptoms
over a period of 60 days post-inoculation. Corm, pseudostem and leaf samples from symptomatic and asymptomatic
plants were aseptically sampled at the end of the experiment and the presence of Xcm conrmed on agar medium
(Mwangi et al. 2007) and using PCR (Adikini et al. 2011).
Pseudostem samples were taken from 10 cm above
the collar region; leaf samples included the leaf petiole
and lamina; while the entire corm was sampled. About
3 g of each sample was macerated with sterile mortar
and pestle in 5 mL of sterile distilled water. One mL of
the resulting macerate was diluted two times. For each
dilution, 20 L was plated onto three Petri plates containing semi-selective YPGA medium and incubated at
28C for 72 h. Characteristic Xcm colonies were
counted and the mean colony forming units (cfu) g1
of plant tissue for the triplicate plates was computed as
indicated below:

Number of cfu  dilution factor  amount plated

grams of plant tissue

vils were removed and Petri dishes washed with 3 mL of

PBS-T by shaking the covered Petri dishes for 5 min at
100 rpm on a mechanical shaker. One mL of the resulting suspension was plated and data captured as previously described in the section(s) above.
Xcm transmission by weevils fed on Xcm oozing corms
This experiment was conducted to determine the potential
of weevils maintained on Xcm oozing corms to transmit
Xcm to healthy potted plantlets. Four different banana
cultivars: Kayinja (Musa ABB), Mpologoma (Musa
EA-AAA), Nakitembe (EA-AAA) and Musakala (EAAAA) were used in this study. A total of 30 plants per
cultivar were each planted in 20 L buckets containing
sterilized soil and arranged in a completely randomized
design. Plants were grown for 3 months prior to treatment
application with continued weeding and watering as

Results
Detection of Xcm in weevils captured from Xcm infected
plants
Xanthomonas campestris pv. musacearum was detected on
both the external and internal body parts of the weevils
captured from banana elds with plants showing characteristic banana Xanthomonas wilt symptoms (Table 1).
Signicant differences (P < 0.001) in Xcm incidence in
the banana weevils were observed between the two cultivar
types, and the external and internal weevil surfaces/organs.
For example, more weevils with Xcm were observed from
Mbwazirume compared with Kayinja elds (Table 1).
Similarly, there was a higher Xcm incidence on the weevil
body surfaces compared with the internal body surfaces/
organs (Table 1). The Xcm-like colonies from both cultivar
types and body parts were conrmed to be positive for Xcm
with PCR (Fig. 1).

Transmission of Xanthomonas by the banana weevil

431

Table 1. Proportion of weevils that had Xcm on the external and internal body surfaces captured from Mbwazirume
and Kayinja elds with banana Xanthomonas wilt symptomatic plants.
Proportion of weevils with Xcm

Field of Mbwazirume (n = 68)

Field of Kayinja (n = 68)
Lsd
Cv%

External surface (%)

Internal organs (%)

Total (external and internal) (%)

55.9 b
41.9 a
4.69***
66.6

45.4 b
17.7 a
4.89***
107.6

66.4 b
50 a
5.28***
63.1

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*** denotes highly signicant differences (P 0.001) between the treatments within each cultivar. Values followed by the same letter
within columns are not signicantly different at 5% LSD.

Fig. 1 Agarose gel photo of PCR amplication of Xcm isolates from different studies. Lane 1 to 5: denotes DNA fragments of Xcm isolates
from weevils captured from Kayinja and Mbwazirume elds; Lane 6 to 9: DNA fragments of Xcm isolates from Kayinja, Musakala,
Nakitembe and Mpologoma, respectively, inoculated with weevils previously fed on Xcm oozing corms; and Lane 10 to 12: DNA
fragments of Xcm isolates from weevil head (including mouth parts), thorax and abdomen. M is a 1kb ladder.

Occurrence of Xcm on the weevil body surface, internal

body parts and faecal matter
Xanthomonas campestris pv. musacearum was isolated
from the external body surface, internal parts (i.e. the
mouth, thorax and abdomen) and the faecal matter of the
weevils that had been maintained on Xcm oozing corms

(Fig. 2a, b). These colonies were conrmed by PCR

using Xcm specic primers (Fig. 1). Signicant differences (P < 0.001) were observed in Xcm incidence and
colony forming units (cfu mL1) between the external
and internal body parts and faecal matter of the weevils
(Fig. 2a, b). Signicantly more weevils had Xcm on their
external body surfaces. The mouth parts ranked second

Fig. 2 Xanthomonas campestris pv. musacearum incidence (a) and bacterial load (CFU mL1) (b) on the body surface, within the internal
organs and the faecal matter of the banana weevil.

E. Were et al.

432

in Xcm incidence and cfu mL1; incidence and cfu mL1

declined along the gut from the mouth parts to the abdomen, with the least observed in the faecal matter. No
signicant differences (P < 0.05) were observed between
the number of weevils with Xcm in the abdomen and
faecal matter (Fig. 2a, b).

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Transmission of Xcm to healthy plants by weevils

Weevils previously fed on Xcm oozing corms were able
to infect healthy 3 month old potted banana plantlets.
The rst symptomatic plants were recorded 36 days postinoculation on positive control plants. The plants that did
not receive any weevils remained healthy for the duration of the experiment and no Xcm was isolated from
them (Table 2). Mpologoma, Musakala and
Nakitembe plants subjected to the different weevil
treatments showed symptoms such as death of tissues
around the point of inoculation, wilting of leaves and
death of the entire plant; while Kayinja remained
asymptomatic for the duration of the experiment
Table 2. Presence and absence of banana Xanthomonas wilt characteristic symptoms on plants treated with 1 weevil (T1), 5 weevils (T5) and 10 weevils (T10) 60 days post-inoculation. Positive
controls were inoculated with known Xcm isolate while the negative controls did not receive any treatment.
Cultivar
Kayinja
Mpologoma
Musakala
Nakitembe
CV%
LSD (5%)

Negative
0a
0a
0a
0a
38.9
0.2***

Positive

T1

T5

T10

100a
100a
100a
100a

0a
33b
67c
67c

0a
100b
100b
100b

0a
67b
100c
100c

*** denotes highly signicant differences (P 0.001) in the different

columns between the treatments within each cultivar. Values followed by
the same letter within columns are not signicantly different at 5% LSD.

(Table 2). Plants that received treatment T10 showed

symptoms earlier than those that received T5 and T1.
PCR analysis of isolates of Xcm from representative
plants from each cultivar conrmed presence of Xcm,
even in Kayinja plants that were asymptomatic (Fig. 1).
Signicant differences (P < 0.001) were also observed
in Xcm incidence and cfu g1 in the different treatments
as well as interaction between treatment and banana cultivar. Incidence was inconsistent and did not correlate to
the number of weevils released to the plants, except for
Kayinja (Fig. 3a). Similar trends were observed in cfu
g1 for each cultivar (Fig. 3b). The highest number of cfu
g1 were observed in Kayinja, Mpologoma and
Musakala for treatment levels T10, T5 and T1, respectively (Fig. 3b). The least colony forming units were
observed in Mpologoma (T10) and Nakitembe (T10).
Trends in cfu g1 were similar in Mpologoma and
Musakala with the highest cfu g1 in plants treated
with T1 and the least in plants treated with T10
(Fig. 3b). In Kayinja, plants treated with T10 had the
highest cfu g1 while those treated with T1 had the least;
however, for Nakitembe, plants treated with T5 had the
highest number of cfu g1 while those treated with T10
had the least number of cfu g1 (Fig. 3b).

Discussion
This study determined the potential of banana weevils to
act as vectors for Xcm. Presently, Xcm has been conrmed to be transmitted by insects such as bees under
eld conditions (Tinzaara et al. 2006). Meki et al. (2010)
conrmed the indirect role of nematodes in soilborne
Xcm transmission through nematode-inicted root
damage in banana. In this study, Xcm was recovered
from within the internal weevil organs as well as the
outer surface of weevils captured from elds with diseased plants. Similarly, Xcm was recovered from the

Fig. 3 Xanthomonas campestris pv. musacearum (Xcm) incidence (a) and bacterial load (CFU g1) (b) for the different cultivars treated with
1 weevil (T1), 5 weevils (T5) and 10 weevils (T10) previously fed on Xcm oozing corms 60 days post-inoculation.

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Transmission of Xanthomonas by the banana weevil

outer surface, internal organs and faecal matter of weevils that were fed on Xcm oozing corms in the laboratory. Windels et al. (1976) reported contamination of
corn seed by picnic beetles (Glischrochilus quadrisignatus) (McCoy & Brindley 1961; Luckmann 1963) carrying Fusarium oxysporum (Windels & Windels 1974)
internally and externally. The presence of Xcm in the
different body parts of the banana weevil and its subsequent occurrence in faecal matter raises questions on
banana Xanthomonas wilt spread and management. For
example, if Xcm can persist through the digestive processes in the insect gut, weevil foraging on infected plant
material could potentially transmit Xcm to healthy
banana plants while foraging or ovipositing eggs.
Weevil eggs are usually laid in the crown of the rhizome
and pseudostem base (Abera et al. 1999). Females oviposit their eggs in small crevices chewed in the plant
tissue (Beccari 1967), thus creating an opportunity to
infect the plant with Xcm.
Under the current Xanthomonas wilt management
practices, farmers cut and chop into pieces diseased
plants that often release excessive Xcm ooze. Weevils
are attracted to volatiles emanating from cut banana
pseudostems, rhizomes and suckers (Gold & Messiaen
2000; Gold et al. 2002) and will thus burrow under these
Xcm oozing corm or pseudostem pieces. When these
pseudostems desiccate, or during oviposition and feeding, these weevils could move and spread bacteria to
infect healthy plants within the eld. In pot trials to
prove this, weevils previously fed on Xcm oozing
corms successfully infected healthy banana plantlets,
irrespective of the number of weevils. These results
suggest that the control of banana weevils is equally
important to the management of banana Xanthomonas
wilt.
Banana weevils are able to move between farms (Gold
& Bagabe 1997; Gold et al. 2002). Flight among adult
banana weevils is rare and adults mainly move by crawling at night with estimated distances of 10 m over
several months (Gold et al. 1999), with only a small
proportion moving beyond 25 m in 6 months (Gold &
Messiaen 2000). For example, Gold et al. (1999) found
only less than 3% of marked weevils in a plot other than
that of their release after 3 years in a markrecapture
experiment. However, similar experiments reported 60%
of marked weevils to have moved more than 10 m over a
2 week period (Gold et al. 2001). Weevil movement is
inuenced by both host plant volatiles and their aggregation pheromone (Tinzaara et al. 2003), although the
distance over which they can sense the volatiles and
orient towards the host plant in the eld is unknown

433
(Gold et al. 1999). These studies suggest that the role
of the banana weevil as a vector of Xcm could be
inuenced by various factors that could include but are
not limited to: the predominant cultivar and management
practices and agro-ecological conditions. From our
results, we observed higher Xcm incidence and cfu
mL1 on weevils captured from Mbwazirume elds
compared with those from Kayinja elds. This could
be attributed to the high attractiveness of Mbwazirume
to the banana weevil. Cultivar preferences associated
with corm hardiness and chemical characteristics (antibiosis) are responsible for weevil preferences to banana
cultivars (Gold et al. 1993). For example, Kiggundu
(2000) showed higher weevil damage in Mbwazirume
compared with Kayinja. However, fewer cfu g1 were
recovered from Mbwazirume and the other highland
bananas compared with Kayinja in the pot trials. This
inconsistency may be due to differential colonization of
Xcm on weevils. Also, it can be attributed to the fact that
samples used for Xcm isolations in the highland cultivars
had full-blown symptoms and had started to senesce
whereas Kayinja samples were fresh though symptomless. It has been observed that recovery of Xcm from
plants with full-blown symptoms and at the point of
senescence is inhibited by the high population of saprophytic microbes (Were Evans, personal communication).
This study suggests that weevils can play a role as
vectors of Xcm and hence the need to incorporate weevil
management in the integrated disease management of
banana Xanthomonas wilt.

Acknowledgements
Special thanks are owed to the International Institute
of Tropical Agriculture (IITA) and Association for
Strengthening Agricultural Research in East and Central
Africa (ASARECA) for funding all activities of this study.
Many thanks to Francis Ssebulime and Ronald Senabulya
for their valuable contribution to this work.

Funding
This work was supported by the International Institute of
Tropical Agriculture Centre code 5296.

References
Abera AMK, Gold CS, Kyamanywa S. 1999. Timing and distribution
of attack by the banana weevil (Coleoptera: Curculionidae) in East
African highland banana (Musa spp.). Fla Entomol. 82:631641.

Downloaded by [Evans Were] at 10:56 15 February 2016

E. Were et al.
Adikini S, Tripathi L, Beed F, Tusiime G, Magembe EM, Kim DJ.
2011. Development of a specic molecular tool for detecting
Xanthomonas campestris pv. musacearum. Plant Pathol. 60:443452.
Beccari F. 1967. Contributo alla conoscenza del Cosmopolites sordidus
(Germ.) (Coleopt., Curcul.). Riv Agr Subtrop Trop. 61:131150.
Budenberg WJ, Ndiege JO, Karago FW, Hansson BS. 1993.
Behavioural and electro-physiological responses of the banana weevil
Cosmopolites sordidus to host plant volatiles. J Chem Ecol. 19:267277.
Castrillon C. 2000. Distribution de las Especies de picudo del platano
Evalucion de sus Entomopatogenous Nativos en el Departemento de
Risaralda CORPOICA. Manizales (Columbia). Manizales. 13.
FAO. 2013 Mar. Core production data. Rome (Italy): FAOSTAT, Food and
Agriculture Organization of the United Nations. http://faostat.fao.org/
site/340/default.aspx
Gold CS, Bagabe MI. 1997. Banana weevil, Cosmopolites sordidus
Germar (Coleoptera: Curculionidae), infestations of cooking- and beerbananas in adjacent plantations in Uganda. Afr Entomol. 5:103108.
Gold CS, Kagezi G, Nemeye P, Ragama P. 2002. Density effects of the
banana weevil Cosmopolites sordidus (Germar) on its oviposition performance and egg and larval survivorship. Insect Sci Appl. 22:205213.
Gold CS, Messiaen S. 2000. The banana weevil Cosmopolites sordidus.
Musa Pest Fact Sheet No 4. Montpellier (France): INIBAP.
Gold CS, Ogenga-Latigo MW, Tushemereirwe W, Kashaija I,
Nankinga CM. 1993. Farmer perceptions of banana pest constraints in
Uganda. In: Gold CS, Gemmil B, editors. Results from a rapid rural
appraisal. Biological and integrated control of highland banana and plantain, Proceeding of Research Co-ordination meeting. Ibadan (Nigeria);
International Institute of Tropical Agriculture (IITA). p. 325.
Gold CS, Pena JE, Karamura EB. 2001. Biology and integrated pest
management for the banana weevil Cosmopolites sordidus (Germar)
(Coleoptera: Curculionidae). Int Pest Manag Rev. 6:79155.
Gold CS, Rukazambuga NDTM, Karamura EB, Nemeye P, Night G.
1999. Recent advances in banana weevil biology, population dynamics
and pest status with emphasis on East Africa. In: Frison EA, Gold CS,
Karamura EB, Sikora RA, editors. Mobilizing IPM for sustainable
banana production in Africa. Montpellier (France): INIBAP; p. 3550.
Kiggundu A. 2000. Host-plant interactions and resistance mechanisms to
banana weevil Cosmopolites sordidus (Germar) in Ugandan Musa germplasm [M.Sc. Thesis]. Bloemfontain (South Africa): University of the
Orange Free State.
Luckmann WH. 1963. Observations on the biology and control of
Glischrochilus quadrisignatus. J Econ Entomol. 56:681686.
Mahuku GS. 2004. A simple extraction method suitable for PCR-based
analysis of plant, fungal and bacterial DNA. Plant Mol Bio Rep. 22:7181.
McCoy CE, Brindley TA. 1961. Biology of the four-spotted fungus
beetle, Glischrochilus quadrisignatus, and its effect on European corn
borer populations. Plant Mol Bio Rep. 54:713717.

434
Meki S, Addis T, Mekonen S, De Waele D, Blomme G. 2010.
Nematode infection predisposes banana to soil-borne Xanthomonas
campestris pv. musacearum transmission. Tree For Sci Biotech. 4:63
64.
Messiaen S. 2002. Components of a strategy for the integrated management of the banana weevil Cosmopolites sordidus (Germar)
(Coleoptera: Curculionidae). Dissertationes de Agriculture No. 540
[PhD thesis]. Belgium: Faculty of Agricultural and Applied Biological
Sciences, Catholic University Leuven.
Mwangi M, Mwebaze M, Bandyopadhyay R, Aritua V, Eden-Green
S, Tushemereirwe WK, Smith J. 2007. Development of a semiselective medium for isolating Xanthomonas campestris pv. musacaerum from infected vectors, infected plant materials and soil. Plant
Pathol. 56:383390.
Sharrock S, Frison E. 1999. Musa production around the world-trends,
varieties and regional importance. In: Networking Banana and Plantain:
INIBAP Annual Report 1998. International Network for the
Improvement of Banana and Plantain, Montepellier, France, pp 4247.
Tinzaara W, Dicke M, Van Huis A, Van Loon JJA, Gold CS. 2003.
Different bioassays for investigating orientation responses of the banana
weevil, Cosmopolites sordidus, show additive effects of host plant
volatiles and a synthetic male- produced aggregation pheromone.
Entom Exp Appl. 106:169175.
Tinzaara W, Gold CS, Ssekiwoko F, Tushemereirwe WK,
Bandyopadhyay R, Abera A, Eden-Green SJ. 2006. Role of insects
in the transmission of banana bacterial wilt. Banana bacterial wilt in
Uganda: a disease that threatens livelihoods. Afr Crop Sci J. 14:105
110.
Tushemereirwe WK, Kangire A, Ssekiwoko F. 2004. First report of
Xanthomonas campestris pv. musacearum on banana in Uganda. Plant
Pathol. 53:802.
Tushemereirwe WK, Okaasai O, Kubiriba J, Nankinga C, Muhangi
J, Odoi N, Opio F. 2006. Status of banana bacterial wilt in Uganda.
Special issue. Banana bacterial wilt in Uganda: a disease that threatens
livelihoods. Afr Crop Sci J. 14:7382.
Van Asten PJA, Gold CS, Wendt J, De Waele D, Okech SHO, Ssali
H, Tushmereirwe WK. 2004. The contribution of soil quality to yield
and its relation with other banana yield loss factors in Uganda. In:
Blomme G, Gold CS, Karamura E, editors. Proceedings of the
Workshop held on farmer participatory testing of IPM options for
sustainable banana production in eastern Africa. Montpellier: IPGRI;
p. 100115.
Windels CE, Mark B, Kommedahl T. 1976. Association of Fusarium
species with picnic beetles on corn ears. Phytopathology. 66:328331.
Windels CE, Windels MB. 1974. Nitidulid beetles as vectors for
Fusarium species on corn. Annu Proc Am Phytopathol Soc. 1:131
(Abstr).