International Journal of Pest Management

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Evidence of fungal structures in the mandible and

elytra of Hypocryphalus mangiferae collected from
mango trees showing symptoms of mango sudden
decline disease

Rogério Machado Pereira, Gustavo Ferreira Martins, Ricardo Siqueira da

Silva, Tarcísio Visintin da Silva Galdino, Dalton de Oliveira Ferreira, Carlos
Alberto Hector Flechtmann & Marcelo Coutinho Picanço

To cite this article: Rogério Machado Pereira, Gustavo Ferreira Martins, Ricardo Siqueira da Silva,
Tarcísio Visintin da Silva Galdino, Dalton de Oliveira Ferreira, Carlos Alberto Hector Flechtmann
& Marcelo Coutinho Picanço (2020): Evidence of fungal structures in the mandible and elytra of
Hypocryphalus�mangiferae collected from mango trees showing symptoms of mango sudden
decline disease, International Journal of Pest Management

To link to this article: https://doi.org/10.1080/09670874.2020.1775324

Published online: 10 Jun 2020.

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INTERNATIONAL JOURNAL OF PEST MANAGEMENT
https://doi.org/10.1080/09670874.2020.1775324

Evidence of fungal structures in the mandible and elytra of Hypocryphalus

mangiferae collected from mango trees showing symptoms of mango
sudden decline disease
Rog
erio Machado Pereiraa, Gustavo Ferreira Martinsb, Ricardo Siqueira da Silvac, Tarc
ısio Visintin da
Silva Galdinod, Dalton de Oliveira Ferreirae, Carlos Alberto Hector Flechtmannf and
Marcelo Coutinho Picançod
a
Departamento de Bioqu
ımica e Biologia Molecular, Centro Universit
ario de Mineiros – UNIFIMES, Mineiros, GO, Brazil;
b
Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil; cDepartamento de Agronomia,
Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Diamantina, MG, Brazil; dDepartamento de Entomologia,
Universidade Federal de Viçosa, MG, Brazil; eDepartamento de Bioqu
ımica e Biologia Molecular, Universidade Federal de Viçosa,
Viçosa, MG, Brazil; fDepartamento de Fitossanidade, Engenharia Rural e Solos, Universidade Estadual Paulista, Ilha Solteira,
SP, Brazil

ABSTRACT ARTICLE HISTORY

Ceratocystis fimbriata plays a significant role in the initiation of the tree decline disease in Received 2 February 2019
mango (Mangifera indica L.), which can kill the plant. The C. fimbriata infection is linked with Accepted 23 May 2020
the presence of the beetle Hypocryphalus mangiferae (Curculionidae: Scolytinae). However, it
KEYWORDS
is unknown how H. mangiferae aids in the transport of the fungus between mango trees.
Insect-fungus associations;
The present study aimed to investigate the external surface of H. mangiferae adults using mango sudden decline
scanning electron microscopy to detect signs of the fungus. Fungal structures were found to disease; mango bark beetle;
adhere to the mandible and elytra of H. mangiferae, implying that H. mangiferae, while mov- fungal structures; insect
ing between mango trees, may spread the fungus through contact. Our findings may be dissemination
useful in developing strategies to minimize the economic impact caused by diseases
through the control of beetles in mango orchards.

1. Introduction montium to Pinus halepensis trees (Vissa and

Fungi are ecologically the most diverse group of
microorganisms and can be beneficial or harmful to
plants (Ellis et al. 2008). Some fungi are able to col-
onize plants using different strategies, which either
benefit (beneficial groups) or harm (pathogens) the
plant. Infection of agricultural plants by pathogenic
fungi can lead to a 100% loss in the production of
commercial crops. Mango Sudden Decline (MSD) is
a fungal disease, which spreads predominantly
through vectors (e.g., insects) (Boomsma et al.
2014). Spread of plant diseases by insect vectors are
common in nature (Hofsetter and Moser, 2014).
Arthropods from the families Curculionidae,
Scolytinae (ambrosia and bark beetles), and
Platypodinae (ambrosia beetles), spread pathogenic
fungi in the families Ophiostomatales, Microascales,
and Hypocreales among species of Pinus (Ploetz
et al. 2013; Hofstetter and Moser 2014). The fly
Bradysia impatiens (Diptera: Sciaridae) spreads
Fusarium oxysporum f. sp. radicis-lycopersici among
tomato, and bean plants (Gillespie et al. 1993).
Mites such as Histiostoma ovalis also act as vectors,
carrying Ophiostoma bruneo-ciliatum, and O.
Hofstetter 2017). The study of associations between
arthropods and plant disease is not new (Webber
and Gibbs 1989; Hajek and St. Leger 1994;
Hofstetter and Moser 2014) and numerous studies
have explored how insects transfer fungus (Al
Adawi et al. 2003, 2013; Harrington 2005; Saeed and
Masood 2008; Shahbaz et al. 2009; Cole and Hoch
2013; Boomsma et al. 2014). Current information
suggests that the mandibles pit found in Scolytidae
species acts as a carrier that preserves and protects
fungal structures prior to infection of plants (Six
2003; Hofstetter and Moser 2014). Fungal pathogens
can be also be carried in the gut, or on the wings,
and mouthpart structures of these insects (Malloch
and Blackwell 1993; Hofstetter and Moser 2014).
However, we still have a poor understanding of
these interactions owing to the complexity, and
diversity of the organisms involved.
In mango orchards, the fungus Ceratocystis fim-
briata, (syn; C. manginecans) causes a devastating
disease known as MSD or Mango Wilt (Al Adawi
et al. 2006; Oliveira, Harrington, et al. 2015;
Oliveira, Guimar~aes, et al. 2015; Galdino et al. 2016;

CONTACT Ricardo Siqueira da Silva ricardo.siqueira@ufvjm.edu.br, ricardo.ufvjm@gmail.com

ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 R. M. PEREIRA ET AL.
Figure 1. SEM image of H. mangiferae (A) mouthparts, (B) mandibles with pits, (C) and (D) elytra with attached structures.
Mandibles (m) and pits (mc) are identified. Arrows indicate fungal structures on the elytra and mandible pits.
Hassan and Nazami 2017). MSD can cause as high
as l of 60% loss of total mango production (Al
Adawi et al. 2006; Al Adawi et al. 2003). In severe
cases, the infection can kill a mango tree (Oliveira,
Harrington, et al. 2015). C. fimbriata infection path-
ways are linked to the beetle Hypocryphalus mangi-
ferae (Curculionidae: Scolytinae), also known as the
mango bark beetle. Typically, MSD symptoms begin
to emerge from holes where beetles enter the bark
(Al Adawi et al. 2013; Souza et al. 2013). It could be
postulated that the inocula of the fungus are depos-
ited at the point where the beetle begins to create
the holes on the stem. This further suggests that the
inoculum is carried on the bodies, especially, the
mouthparts of the beetle. However, this possibility
has not been investigated. Therefore, the exact role
that H. mangiferae plays in the transfer of C. fim-
briata spores remains unclear, despite various
reports that discuss the causes and vectors of MSD.
Specifically, it is not well understood which body
parts of the beetle are involved in the transfer of the
fungus. In view of the shortfall in knowledge regard-
ing the mechanism involved in the transfer of
inoculum of C. fimbriata by the bark beetle, this
present study investigates the external surface of H.
mangiferae adults collected from mango orchards
infected with C. fimbriata, to 1) determine which
specialized structures are involved in the transfer of
the fungus, and 2) to detect the presence of fungal
propagules in the powdery sawdust created by
the beetles.
2. Materials and methods
2.1. Mango orchards
The study was carried out in two commercial
mango orchards in Itaocara, Rio de Janeiro, Brazil
(
21 380 14.4000 S,
41 570 19.1200 W and
21
380 11.2200 S,
41 570 5.7000 W). The mango trees were
of the “Espada” variety. Farm sizes were 2.6, and 2.3
hectares, respectively.
2.2. Materials collected
Two different plant parts, namely the trunk and
branches were collected from the infected orchards.

Figure 2. Percent isolation of C. fimbriata from powdery
sawdust and stem tissue samples. Carrot disks without a
sample were used as the control.
Trunks had dimensions of 5 cm  10 cm  3 cm
(width, height, depth) and branches of 5 cm
diameter and length 50 cm were collected. A total
of 50 samples were collected from 50 mango trees
showing MSD symptoms (25 from each orchard). The
collected samples were subsequently packed in brown
paper bags and transported to the Integrated Pest
Management (IPM) laboratory at the Universidade
Federal de Viçosa (UFV), where stem samples
underwent further sectioning in order to capture live
H. mangiferae adults. A total of 20 beetles (10 from
each orchard) were collected and stored at 4  C
in 2.0 ml sterile microcentrifuge tubes containing
a picric acid-formaldehyde fixative (Zamboni fixative).
Powdery sawdust (20 samples, 10 from each orchard)
produced by H. mangiferae activity, were collected and
stored in 2.0 ml sterile microcentrifuge tubes.
2.3. Scanning electron microscopy
Eight beetles out of the 20 collected were selected at
random and subjected to desiccation. These beetles
were washed thrice with 0.1 M PBS (phosphate-
buffered saline, pH 7.2), followed by dehydration with
an ethanol series (70, 80, 95, and 100%) with a 10-min
wash between each. The insects were subjected to
further dehydration using a Critical Point Dryer (Bal-
tec, CPD 030). The number of successfully desiccated
beetles was relatively low (5 samples); therefore,
a discrete analysis of elytra was carried out by separat-
ing them from the intact carcass using pincers.
Subsequently, the insect body and elytra were mounted
on stubs and sputter-coated with gold, FDU 0:10.
Images were captured using an LEO 1430 VP SEM at
the N
ucleo de Microscopia e Microan
alises at UFV.
INTERNATIONAL JOURNAL OF PEST MANAGEMENT 3
2.4. Identification of isolated fungi
C. fimbriata was isolated using carrot insulation
medium (Moller and DeVay 1968). Powdery
sawdust produced by the beetles (200 mg) and stem
tissue (a 1  1  0.1 cm section) with disease symp-
toms, were placed directly on untreated carrot disks
3 cm  2 cm  0.5 cm thick. Materials used to collect
and handle the samples were sterilized between sam-
ples. The carrot disks were placed in Petri dishes
and incubated in a growth chamber (CDG model
347) at 25  C under a 12-hour photoperiod for
10 days. Carrot disks without powdery sawdust
served as a control. The presence of ascospores in
perithecia on the carrot disks indicated the presence
of C. fimbriata in the sawdust and stem tissue.
The carrot disks were examined microscopically
to confirm the identity of C. fimbriata, following
Moller and DeVay (1968).
3. Results
3.1. Fungal structures on the beetles
Fungus was found on a total of 5 beetle specimens.
Presence of fungal structures (e.g., hyphae and
amorphous masses) adhering to the base of the
mandibles of H. mangiferae was evident by SEM
(Figure 1), corresponding to the mandible pits
(Figure 1(a, b)). Fungal structures were found at the
tip, and ventral face (in contact with the body) of
the elytra (Figure 1(c, d)).
3.2. Presence of fungus in the powdery sawdust
and stem tissues
C. fimbriata was found in 7% of sawdust samples
analyzed, and 100% of the stem tissue samples
showed the symptoms of MSD. C. fimbriata was not
found in the healthy tissue samples (Figure 2).
4. Discussion
Thorough analysis of H. mangiferae beetles using
SEM demonstrated that fungal structures were on
the body of the beetles, including their mandible
pits. These observations suggest that H. mangiferae
could act as a potential vector of C. fimbriata, a
fungal pathogen that infects mango trees. Isolations
from the sawdust and trunks of infected trees
yielded ascospores produced in perithecia, character-
istic of C. fimbriata (Moller and DeVay 1968).
Therefore, it can be deduced that the fungal struc-
tures found on H. mangiferae body surfaces are
those of C. fimbriata and therefore the beetle may
play an essential role in spreading C. fimbriata
between mango trees.
4 R. M. PEREIRA ET AL.
The fungal structures found on H. mangiferae’s
body surface could aid in a better understanding of
the spread of MSD in mango orchards, in addition
to a broader understanding of beetle-fungus
interactions. Indeed, an understanding of these
interactions is needed for the development of mod-
els that can predict the dynamics of fungal infec-
tions in mangos trees, including both applied and
theoretical viewpoints. An earlier study by Oliveira
et al. (Oliveira, Harrington, et al. 2015; Oliveira,
Guimar~aes, et al. 2015) confirmed that the fungus
C. fimbrata was responsible for MSD symptoms on
the same trees used in this study, using morpho-
logical characterisation, mating compatibility, and
genetic tests. This therefore gives credence to our
findings that the fungus isolated from the saw dust
was C. fimbriata.
Curbing the spread of C. fimbriata is challenging
once a mango tree is infected (Ellis et al. 2008).
Therefore, strategies to prevent damage due to MSD
must restrict the entry of the fungus to the orchard.
Our findings, which showed that fungal structures
can attach to the body of H. mangiferae, imply that
monitoring and controlling these beetles could be
done as a basic approach to limit the spread of the
fungus. Sampling plans for these species of fungus
and beetle are not yet developed, but other research-
ers indicate their potential for being established in
new areas (Galdino et al. 2016, 2017).
The fungus C. fimbriata was isolated from 7% of
the samples analyzed. It has been reported that a
single mango tree may have up to 6,000 beetles bor-
ing into the stem, meaning 400 of these beetles
could be carrying the fungus. This statistic demon-
strates how active fungal infection could be spread
by beetles. Thus, an integrated disease management
program in mango should consider the population
of beetles present in the mango orchard.
SEM proved to be a useful tool for analyzing the
presence, and association, of fungal structures on
beetles, providing insight into how the disease may
spread in mango orchards. The present study out-
lines the involvement of beetles in disease transmis-
sion, but there is an urgent need for further studies
regarding the transmission of the fungus within the
beetle body.
Acknowledgements
The authors would like to thank VALE, VALE Oman, the
National Council for Scientific and Technological
Development (Conselho Nacional de Desenvolvimento
Cient
ıfico e Tecnol

ogico – CNPq), the Brazilian Federal
Agency for the Support and Evaluation of Graduate
Education (Coordenaç~ao de Aperfeiçoamento de Pessoal
de Ensino Superior – CAPES) Finance Code 001, the
Minas Gerais State Research Foundation (Fundaç~ao de
Amparo a Pesquisa do Estado de Minas Gerais –
FAPEMIG) for financially supporting this research and
N
ucleo de Microscopia e Microan
alises the Universidade
Federal de Viçosa (NMM/UFV) for technical assistance.
Disclosure statement
The authors declare that they have no conflict of interest.
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