Lasiodiplodia theobromae as a causal pathogen of leaf blight, stem canker, and pod rot of Theobroma cacao in Malaysia

Abstract

Symptoms of leaf blight, stem canker, and pod rot were observed on T. cacao during a series of samplings conducted in several states of Malaysia from September 2018 to March 2019. The identity of the pathogen that was responsible for the diseases was determined using morphological characteristics, DNA sequences, and phylogenetic analyses of multiple genes, namely, internal transcribed spacer (ITS), elongation translation factor 1-alpha (tef1-α), β-tubulin (tub2), and RNA polymerase subunit II (rpb2). A total of 57 isolates recovered from diseased leaves of T. cacao (13 isolates), stems (20 isolates), and pods (24 isolates) showed morphological features that resembled Lasiodiplodia sp. The identity of the isolates was further determined up to the species level by comparing DNA sequences and phylogenetic analyses of multiple genes. The phylogenetic analysis of the combined dataset of ITS, tef1-α, tub2, and rpb2 elucidated that all of the isolates obtained were Lasiodiplodia theobromae as supported by 97% bootstrap value. The results of pathogenicity tests revealed L. theobromae as the causal pathogen of leaf blight, stem canker, and pod rot of T. cacao.

Full text

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Lasiodiplodia theobromae
as a causal pathogen of leaf
blight, stem canker, and pod rot
of Theobroma cacao in Malaysia
Abd Rahim Huda‑Shakirah1, Nik Mohd Izham Mohamed Nor1, Latiah Zakaria1,
Yin‑Hui Leong2 & Masratul Hawa Mohd1*
Symptoms of leaf blight, stem canker, and pod rot were observed on T. cacao during a series
of samplings conducted in several states of Malaysia from September 2018 to March 2019. The
identity of the pathogen that was responsible for the diseases was determined using morphological
characteristics, DNA sequences, and phylogenetic analyses of multiple genes, namely, internal
transcribed spacer (ITS), elongation translation factor 1‑alpha (tef1-α), β‑tubulin (tub2), and RNA
polymerase subunit II (rpb2). A total of 57 isolates recovered from diseased leaves of T. cacao (13
isolates), stems (20 isolates), and pods (24 isolates) showed morphological features that resembled
Lasiodiplodia sp. The identity of the isolates was further determined up to the species level by
comparing DNA sequences and phylogenetic analyses of multiple genes. The phylogenetic analysis of
the combined dataset of ITS, tef1-α, tub2, and rpb2 elucidated that all of the isolates obtained were
Lasiodiplodia theobromae as supported by 97% bootstrap value. The results of pathogenicity tests
revealed L. theobromae as the causal pathogen of leaf blight, stem canker, and pod rot of T. cacao.
e cocoa tree (eobroma cacao) is an evergreen shrub that is recognized by several names, including kakaw,
pokok coklat, chocolate, cacao, koko, criollo, cacaoyer, and kakao1. Previously, T. cacao was classied under
Sterculiaceae family, before being reclassied as a member of Malvaceae. It is originated in the Neotropical
rainforest, particularly in the Amazon basin and on the Guyana plateau2–4. e word eobroma means “Food
of the Gods,” whereas cacao comes from the Mayans and Aztec languages, Kakaw and Cacahuatl, respectively5,6.
Furthermore, T. cacao is the recognized species among the 22 eobroma species that is commonly planted
beyond its natural range and have an economic value1,6. Besides T. cacoa, the other species of eobroma also
have economic value such as T. grandiorum in South America and T. bicolor in Mexico and Central America6.
Clone seedling is preferred for plantation over hybrid seedling in almost all cocoa-producing countries because
it will produce the same tree morphology, pod, and bean characteristics as the parent tree, where the clone tree
has greater pod bearing capacities, bigger and more uniform beans, richer butter content, withstand to pest and
pathogen attacks, and adaptable to a wide range of agro-climatic conditions1,7. e continued advancement of
Malaysia’s cocoa industry in the late 1970s and early 1980s resulted in the founding of the Malaysian Cocoa Board
(MCB) in 1989, which is overseen by the Ministry of Plantation Industries and Commodities. e Board’s goal
was to grow Malaysia’s cocoa industry so that it could be incorporated in the global market, as well as to boost
the quality and performance of cocoa bean and downstream production8. Malaysia is now the leading country
in the cocoa grinding industry8.
In addition, cocoa and its products have various nutritional values owing to their rich amounts of alkaloids,
cardiac glycosides, catechin, enantiomer, epicatechin, avanol, methylxanthines, procyanidin B2, saponin, tan-
nins, and terpenoids9. Moreover, cocoa has several biological benets, including high antioxidant activity, blood
pressure reduction, anticancer activity, stress and depression reduction, reduced risk of heart attack and stroke,
cholesterol control, antiplatelet eect, and anti-inammatory activity10–14.
eobroma cacao tree, similar to any other Malvaceae plants, has been shown to be fungus-prone. Among
the most important diseases aecting cacao in Malaysia are black pod rot, canker, and vascular streak dieback
(VSD), which aect the pod; trunk and stem; leaves and stems of the cacao tree, respectively1. Furthermore,
several previous studies on the diseases of T. cacao caused by fungal and fungal-like pathogens have been reported
OPEN
1School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. 2National Poison
Centre, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. *email: masratulhawa@usm.my
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worldwide namely, Ceratobasidium theobromae15, Colletotrichum gloeosporioides6, Colletotrichum siamense16,17,
Colletotrichum theobromicola18, Colletotrichum tropicale17, Lasiodiplodia brasiliensis19, Lasiodiplodia pseudothe-
obromae17, Lasiodiplodia theobromae6,19–25, Moniliophthora perniciosa26, Moniliophthora roreri27, Neofusicoccum
parvum28, Phytophthora palmivora6,25,29, and Phytophthora megakarya4,29.
In a series of samplings conducted from September 2018 to March 2019, the occurrences of leaf blight,
stem canker, and pod rots of T. cacao were observed in cocoa plantations in several states of Malaysia. From
observations during the sampling revealed the disease incidences of leaf blight, stem canker, and pod rots in
cocoa plantations were 15%, 20%, and 25%, respectively, which may reduce cocoa production. e diseased
samples were gathered and returned for further observation. erefore, the present study sought to nd the
causative agent of leaf blight, stem canker, and pod rot of T. cacao in Malaysia using morphological, molecular,
and pathogenicity analyses.
Results
Fungal isolation and morphological identication. In total, 57 fungal isolates were retrieved from dis-
eased leaves of T. cacao (13 isolates), stems (20 isolates), and pods (24 isolates). On PDA, the fungal isolates pro-
duced dense and fast-growing mycelia, white to pale greenish-gray colony and eventually becoming dark grayish
(Fig.1A). e pigmentation ranged from dark gray to black (Fig.1B). e conidiomata were solitary, globose
to subglobose, uniloculate, black, surrounded by dense grayish mycelia, and 3.32 ± 0.47 × 3.10mm ± 0.27mm
(mean ± standard deviation (SD)) (length (L) × width (W)) in size (Fig. 1C). e conidia were observed as
immature and mature conidia. Both immature and mature conidia were subovoid to ellipsoid-ovoid in shape,
with a broadly rounded apex and a tapering to the truncated base. e immature conidia were initially dou-
ble layered, hyaline, unicellular, and 25.0 ± 1.06 × 13.0 µm ± 0.48 µm (mean ± SD) (L × W) in size (Fig. 1D).
e mature conidia appeared light to dark brown color with typical striate formation, one-septate, and
25.7 ± 1.73 × 13.1µm ± 0.82µm (mean ± SD) (L × W) in size (Fig.1E). e conidiogenous cells were cylindrical,
hyaline, thin walled, holoblastic, and smooth. e structure of the paraphyses was aseptate and septate, with
rounded apex, hyaline, and cylindrical (Fig.1F). Based on the characterization of the morphological features
of the fungal isolates, it was tentatively identied as Lasiodiplodia sp., which is coherent with the morphology
described by Alves etal.30 and Phillips etal.31.
Figure1. Morphological characteristics of Lasiodiplodia sp. recovered from diseased leaves, stem, and pods
of eobroma cacao. (A) Upper view of the colony appearance, (B) Reverse view colony appearance, (C)
Conidiomata, (D) Immature conidia, (E) Mature conidia, (F) Conidiogenous cells and paraphyses. Scale bars:
(C) = 1mm; (D–F) = 50µm.
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Molecular identication and phylogenetic analysis. Molecular analysis of the sequences of ITS,
tef1-α, tub2, and rpb2 claried the species identication of all the 57 isolates of Lasiodiplodia sp. recovered from
T. cacao. BLAST searches in the GenBank database revealed that the isolates showed 98–100% sequence homol-
ogy to the KY473071 (ITS), JX464026 (tef1-α), EU673110 (tub2), and MT592333 (rpb2) of L. theobromae. A
multi-locus analysis was performed to explicate the phylogenetic positions of these L. theobromae isolates. To
construct the phylogenetic tree, the sequences of the isolates from the present study (57 isolates of L. theobro-
mae) were aligned with 38 reference isolates of Lasiodiplodia species and one outgroup taxon (Botryosphaeria
dothidea). Phylogenetic analysis revealed that the topologies of the ML trees generated from individual and con-
catenated genes (ITS, tef1-α, tub2, and rpb2) were similar (Figs.S1a–d and 2). e ML tree constructed from the
concatenated sequences conrmed that the phylogenetic positions of the 57 isolates from T. cacao were clustered
with the reference isolates of L. theobromae, supported by 97% bootstrap value (Fig.2). As a result, all the present
isolates were veried as L. theobromae by virtue of molecular identication and phylogenetic analysis.
Pathogenicity test. e pathogenicity analysis of 13, 20, and 24 fungal isolates on healthy leaves, stems,
and pods of T. cacao resulted in the production of typical symptoms of blight, canker, and rot, respectively as
observed in the elds (Fig.3A,G,R). ere were no visible symptoms produced on control points of leaves, stems,
and pods (Fig.3B,H,S).
Aer 4days of inoculation, the fungal inoculated leaves exhibited small irregular black lesions bounded
by yellow halos (Fig.3C). e lesions and yellowing areas enlarged gradually during the incubation period
(Fig.3D,E). Conidiomata formed on the inoculation site (Fig.3F). e lesion areas produced ranged from 3.0
to 4.6 cm2 (Table1). ere was no signicant dierence of lesion areas recorded among the tested isolates.
e fungal inoculated stems developed black necrotic lesions within the rst to the third week of inoculation
(Fig.3I–K). Aer 4weeks, the lesions extended longitudinally from the inoculation sites (Fig.3L). e incision
of the stem inoculated point displayed a reddish-brown to black necrotic lesion (Fig.3M,N). Formation of gum-
mosis on the necrotic lesion was also observed (Fig.3O). Vertical and transverse sections of control and fungal
inoculated stems showed symptomless and dark brown to black necrotic lesions, respectively (Fig.3P,Q). ere
were signicant dierences of lesion areas produced on the L. theobromae inoculated stems that ranged from
12 to 14 cm2 (Table1).
e fungal inoculated pods showed irregular brown to black lesions aer 5days of incubation (Fig.3T). As
the infection progressed, the lesions expanded and turned darker aer 7days of inoculation (Fig.3U). Aer
12days of inoculation, the lesions continued to expand, and the inoculated pods were completely colonized by
the fungal grayish mycelia (Fig.3V,W). Black conidiomata formed on the fungal inoculated pods (Fig.3X). A
cross-section of fungal inoculated pods showed rotting of the internal tissue (Fig.3Y). e lesion areas ranged
from 46.7 to 50.3 cm2 (Table1). e lesion areas recorded on the fungal inoculated pods were signicantly dif-
ferent compared to the control (Table1).
e repetition of the pathogenicity assessment yielded the same outcomes as the rst analysis. Koch’s postu-
lates were achieved by reisolating the same fungal isolates from the symptomatic inoculated leaves, stems, and
pods of T. cacao and their identities were conrmed through morphological features.
Discussion
e present study identied L. theobromae isolates responsible to cause leaf blight, stem canker, and pod rot of T.
cacao in Malaysia based on the morphological features, sequence comparison, and phylogenetic analysis of four
genes (ITS, tef1-α, tub2, and rpb2). Fungi from genus Lasiodiplodia are cosmopolitan and belong to the Botry-
osphaeriaceae family, and most of the species can be primarily found in tropics and subtropics31–33. e genus
consists of many phytopathogenic fungal species with widespread distribution33. Lasiodiplodia species responsible
to cause over 500 plant diseases, including fruit rot, root rot, collar rot, stem-end rot, dieback, canker, and leaf
necrosis32,34–43. In Malaysia, Lasiodiplodia species have been attributed to various destructive diseases, such as
black rot of kenaf seeds44, leaf blight of Sansevieria trifasciata45, stem end-rot of Mangifera indica46, stem canker
on Jatropha curcas and Acacia mangium47,48, and fruit rot of mango and guava49,50. Apart from that, they can
act as secondary pathogens or endophytes, and they also can become pathogenic in response to a stressor34,36,40.
All the 57 fungal isolates recovered from diseased T. cacao in the present study was tentatively assigned as
Lasiodiplodia sp. based on their macroscopic and microscopic characteristics. According to Hyde etal.51, the
morphological approach has been widely used as the foundation for almost all studies of fungal taxonomy. Slip-
pers and Wingeld34 also stated that Botryosphaeriaceae members are easily recognized from most other fungi
through their colony appearance, aerial mycelium, and pigments, which can aid in the delimitation and rapid
identication. However, due to the signicant overlapping of key morphological characteristics among Lasiodip-
lodia species, clear-cut identication of the Lasiodiplodia isolates in the present study could not be achieved up
to the species level by using traditional morphological descriptions such as conidial shape30,40.
Attributable to unresolve identity of Lasiodiplodia isolates based on morphological characteristics that could
lead to uncertain and misleading results, phylogenetic analysis involving DNA sequences of multiple genes was
applied to delineate species boundaries. Consistent with previous studies that also highlighted the importance
of molecular work in dening Lasiodiplodia species34,39,40,52, the present study used several genes, namely, ITS,
tef1-α, tub2, and rpb2, to explicitly characterize Lasiodiplodia isolates. e ITS region has been proposed and
widely used in fungal taxonomic classication because of its straightforward amplication and it provides a
high probability of successful fungal recognition, with the barcoding dierence between inter- and intraspecic
variations53,54. Nonetheless, the ITS region lacks interspecies variety and may even be vague in the identication
of some fungi, thus the use of additional genes would provide better resolution in the phylogenetic analysis. Other
studies also showed that a single gene is incapable of determining species in the genus Lasiodiplodia, implying that
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Botryosphearia dothid ea CBS11547 6
Lasiodiplodia brasiliensis CBS115447
Lasiodiplodia brasiliensis CMM4015
98 Lasiodip lodia brasiliensis CSM11
NS2F
M3F
M4F
NS7F
NS8F
PP9F
PP11F
J13F
J15F
J16F
M19F
PE20F
PE22F
PP23F
K25F
K27F
S30F
PE31F
PE32F
S34F
S35F
97 PR36F
PR37F
PE39F
K41L
K42L
PR43L
PR44L
PE45L
PE46L
S47L
S48L
S49L
M50L
M51L
NS52L
NS53L
J54S
J55S
J56S
J57S
J58S
J59S
98 NS60S
NS61S
NS62S
M63S
M64S
S65S
S66S
PE67S
PE68S
PP69S
PP70S
PP71S
J72S
51 J73S
Lasiodiplodia theo bromae CBS164.69
Lasiodiplodia theo bromae CBS214.50
Lasiodiplodia theo bromae CMW13490
Lasiodiplodia theo bromae CMM4019
Lasiodiplodia theo bromae CSM57
Lasiodiplodia theo bromae M400
Lasiodiplodia brasiliensis CBS123095
100
99
Lasiodiplodia hormozga nensis CBS12470
9
100 Lasiodiplo dia hormozganensis CBS12470
8
100 Lasiod iplodia mahajangana CBS124925
Lasiodiplodia mahajang ana CBS124926
Lasiodiplodia viticola CBS1 28313
99 Lasiodiplodia viticola CBS128314
100 Lasiod iplodia iraniensis CBS214711
Lasiodiplo dia iraniensis
CB
S124710
91 Lasiodiplo dia iranensis CMW35881
99 Lasiodip lodia euphorbicola CMM3651
Lasiodiplodia euphorbicola CMW33268
Lasiodiplodia euphorbicola CMM3609
Lasiodiplodia citricola CBS1 24707
99 Lasiodiplo dia citricola CBS124706
100
Lasiodiplodia mediterran ea CBS137783
Lasiodiplodia mediterran ea CBS137784
100 Lasiodiplodia lignicola CBS134112
75 Lasiodiplodia lignicola MF
L
U
CC
110656
94 Lasiodiplodia pseudotheo bromae I46
Lasiodiplodia pseudo theobromae CBS116459
100
96 Lasiodiplodia pseudo theobromae
CB
S116460
Lasiodiplodia pseudo theobromae
CB
S130991
Lasiodiplodia crassisp ora CBS118741
Lasiodiplodia crassisp ora CBS125626
Lasiodiplodia crassisp ora CMW33262
98 Lasiodiplodia margar itacea CBS122519
Lasiodiplodia marg aritacea CBS138289
100 Lasiod iplodia margaritacea CBS138290
0.05
Figure2. e maximum likelihood (ML) tree was generated with 1000 bootstrap replications using the
Tamura-3-parameter model. e ML tree is inferred from concatenated sequence dataset of four genes (ITS,
tef1-α tub2, and rpb2). Bootstrap support values greater than 50% are pointed out at the nodes. Isolates in bold
represent isolates in the present study and Botryosphearia dothidea represents the outgroup. e bar indicates
the substitutions number per position.
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Figure3. Pathogenicity of Lasiodiplodia theobromae on leaves, stems, and pods of eobroma cacao. (A) Blighted leaf observed in
the eld, (B) Asymptomatic control inoculated leaf, (C) Irregular black lesions with yellow halo observed aer 4days of inoculation
(D,E) e lesions enlarged aer 6 and 9days of inoculation, respectively, (F) Presence of conidiomata on the diseased area (red arrow),
(G) Cankered stem observed in the eld, (H) Asymptomatic control inoculated stem, (I–K) Black necrotic lesions observed on the
inoculation sites aer 7, 14, and 21days of inoculation, respectively, (L) Black necrotic lesions extending upwards and downwards aer
28days of inoculation, (M) Black sunken lesion on the inoculation site, (N) Incision of the stem inoculated site showed reddish-brown
to black necrotic lesion, (O) Formation of gummosis on the necrotic lesion, (P) Vertical section of control (le) and fungal inoculated
stems (right) showed symptomless and dark brown to black necrotic lesion, respectively, (Q) Transverse section of control (below)
and fungal inoculated stems (above) showed symptomless and necrotic lesion, respectively, (R) Rotted pod observed in the eld
showed external and internal rotting symptoms, (S) Asymptomatic control inoculated pod, (T) Brown to black lesions observed on the
inoculation sites aer 5days of inoculation, (U) e lesions enlarged aer 7days of inoculation (V), e lesion rapidly expanded aer
9days of inoculation, (W) e inoculated pod completely covered by the fungal mycelia aer 12days of inoculation, (X) Presence of
black conidiomata (red circle) on the fungal inoculated pod, (Y) Cross-section of fungal inoculated pod showed rotting of the internal
tissue.
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Isolate code
aLesion area (cm2)
Leaf Stem Pod
K41L 3.3 ± 0.7b b– –
K42L 3.1 ± 0.1b– –
PR43L 3.3 ± 0.7b– –
PR44L 3.7 ± 1.0b– -
PE45L 3.3 ± 0.3b– –
PE46L 3.0 ± 0.3b– –
S47L 4.6 ± 1.2b– –
S48L 4.6 ± 1.2b– –
S49L 3.5 ± 1.0b– –
M50L 3.3 ± 0.4b– –
M51L 3.1 ± 0.3b– –
NS52L 4.0 ± 1.3b– –
NS53L 3.2 ± 0.6b– –
J54S – 14 ± 0d–
J55S – 14 ± 0d–
J56S – 14 ± 0d–
J57S – 14 ± 0d–
J58S – 14 ± 0d–
J59S – 14 ± 0d–
NS60S – 12.3 ± 0c–
NS61S – 12.3 ± 0c–
NS62S – 14 ± 0d–
M63S – 13.1 ± 0cd –
M64S – 13.1 ± 0cd –
S65S – 13.1 ± 0cd –
S66S – 13.1 ± 0cd –
PE67S – 13.1 ± 0cd –
PE68S – 13.1 ± 0cd –
PP69S – 12.0 ± 0c–
PP70S – 12.0 ± 0c–
PP71S – 13.1 ± 0cd –
J72S – 13.1 ± 0cd –
J73S – 13.1 ± 0cd –
NS2F – – 49.8 ± 5.3e
M3F – – 50.3 ± 3.5e
M4F – – 47.9 ± 4.0e
NS7F – – 49.2 ± 3.8e
NS8F – – 48.1 ± 4.4e
PP9F – – 48.0 ± 2.6e
PP11F – – 47.1 ± 6.8e
J13F – – 49.8 ± 7.8e
J15F – – 47.8 ± 10.1e
J16F – – 49.9 ± 7.7e
M19F – – 46.7 ± 8.0e
PE20F – – 48.3 ± 10.6e
PE22F – – 46.7 ± 8.0e
PP23F – – 49.4 ± 4.8e
K25F – – 48.3 ± 5.2e
K27F – – 47.8 ± 8.9e
S30F – – 47.2 ± 7.5e
PE31F – – 50.8 ± 12.6e
PE32F – – 49.2 ± 3.8e
S34F – – 46.8 ± 2.3e
S35F – – 46.2 ± 10.8e
PR36F – – 49.2 ± 3.8e
Continued
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additional genes are required30,55. e tef1-α has become the marker of choice for fungal identication because
of its distinct polymorphisms among similar species and consists of non-orthologous copies of the gene that are
undetected in the genus56. e tub2 is another useful marker for delineating fungal species because it has fewer
obscure aligned regions and less homoplasy across genera57. e rpb2 gene which codes for the second-largest
protein subunit in fungi is a highly preserved single-copy gene54.
According to the results of phylogenetic analysis, it can be inferred that single gene analyses of ITS, tub2,
and rpb2 are unable to resolve the identity of Lasiodiplodia isolates in the present study (Fig.S1a,c,d). ose
phylogenetic trees displayed that L. theobromae was grouped with L. brasiliensis and L. hormozganensis. On the
contrary, phylogenetic analysis of tef1-α sequences was able to dierentiate isolates in the present study with
other species of Lasiodiplodia by clustering them with several reference sequences of L. theobromae from the
GenBank database with only 64% bootstrap value (Fig.S1b). Owing to the fact that single gene analysis could not
accurately identify the Lasiodiplodia isolates in the present study, the combination of ITS, tef1-α, tub2, and rpb2
sequences was used for better characterization. e phylogenetic inferences based on multiple gene sequences
revealed that the present isolates were grouped with L. theobromae with a higher bootstrap value (97%) (Fig.2).
e nding has been proven that phylogenetic analysis based on multigene provided robust resolution with
clear-cut fungal identity. is is in line with the ndings of Cruywagen etal.52.
Lasiodiplodia theobromae was conrmed to be the causal pathogen of leaf blight, stem canker, and pod rot of
T. cacao in Malaysia. In 1895, L. theobromae was rstly described and reported to cause minor charcoal rot on
cocoa in Ecuador31. Besides charcoal rot, L. theobromae was also reported to cause dieback on T. cacao since the
late 1980s20. In Malaysia, documentations of relationship between L. theobromae and T. cacao are still limited.
e present study represents the rst report of leaf blight, stem canker, and pod rot of T. cacao caused by L. theo-
bromae. Several studies have also found the incidence of L. theobromae causing foliar diseases in a wide range
of hosts, including Camellia sinensis42, Catasetum mbriatum58, Cocos nucifera59,60, Kadsura longipedunculata61,
and S. trifasciata45. Moreover, the present study also revealed the ability of L. theobromae isolates to cause stem
canker of T. cacao. Asman etal.24, previously reported L. theobromae as a causal agent of dieback and stem
canker of cocoa by demonstrating internal discoloration with visible brown streaks in the vascular cambium.
Furthermore, L. theobromae has been associated with cocoa dieback in Cameroon, India, and Venezuela19–21. It
also responsible to cause dieback and stem canker on a number of plants, such as American ash (Fraxinus ameri-
cana)62, blueberry bushes (Vaccinium spp.)63, strawberry (Fragaria × ananassa)41, mango (M. indica)64, cashew
(Anacardium occidentale)65, sacha inchi (Plukenetia volubilis)66, Persian lime (Citrus latifolia)67, and grapevine
(Vitis vinifera)68. In addition to infecting the leaf and stem, cocoa pod was also found to be susceptible to L.
theobromae infection by showing rot symptoms. Several studies reported the occurrence of pod rot of T. cacao
caused by L. theobromae6,22,25. Other pathogens were also identied to cause the same disease on the cocoa pod,
namely C. gloeosporioides6, C. siamense17, C. tropicale17, L. pseudotheobromae17, N. parvum25, P. palmivora6,25,29,
and P. megakarya4,29. From the pathogenicity tests, isolates of L. theobromae required wound to initiate infection
and colonization on the host plant. Other studies have found that fungi from Botryosphaeriaceae can invade
plants via endophytic conquest, injuries, seed-to-seedling conquest, contaminated soil, and insect infestation34,36.
In conclusion, the current study emphasized the rst report of L. theobromae as a causal pathogen of leaf
blight, stem canker, and pod rot of T. cacao in Malaysia. e pathogen was identied using morphological fea-
tures supported by multigene DNA sequences and phylogenetic inference. e valid and precise identication
of phytopathogen is critical for quarantine purpose and disease management strategies.
Materials and methods
Collecting samples and isolating fungi. From September 2018 to March 2019, sampling was conducted
during rainy season in several states of Malaysia, including Johor, Kedah, Melaka, Negeri Sembilan, Perak, Perlis,
Pulau Pinang, and Selangor (Fig.4). e sampling sites and sampling activities were approved by the MCB com-
ply with relevant institutional, national, and international guidelines and legislation. During the sampling, 50
blighted leaves, cankered stems, and rotted pods of T. cacao from the Koko Mardi (KM) clone were collected. e
clone was used in the study because of its wide cultivation in Malaysia which showed susceptibility to a number
of fungal diseases. Symptomatic leaves showed blighted symptoms, including circular to irregular blackish lesion
surrounded by a yellow halo. e cankered stems were characterized as irregular blackish lesion, sometimes
accompanied by gummosis on the disease area, expanded longitudinally, and internally became reddish-brown.
e rotted pods were associated with dark brown to blackish lesions on the pods that eventually expanded and
rotted.
Table 1. Lesion area produced on the leaves, stems and pods of eobroma cacao inoculated with
Lasiodiplodia theobromae. a Means ± standard deviation followed by dierent letters are signicantly dierent
(p < 0.05) according to Tukey’s test. b Not applicable.
Isolate code
aLesion area (cm2)
Leaf Stem Pod
PR37F – – 49.6 ± 12.8e
PE39F – – 50.5 ± 7.1e
Control 0a0a0a
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e diseased and healthy margins of samples were cut into small pieces for fungal isolation. e small pieces
of samples were surface-sterilized in 70% ethanol (C2H5OH) and 1% sodium hypochlorite (NaOCl) separately
for 3min. e samples were then rinsed in sterile distilled water three times in succession for 1min each. e
sterilized sample was blotted dry on sterile lter paper, transferred onto potato dextrose agar (PDA), and incu-
bated at 25°C ± 2°C for 3–5days. Pure cultures of fungal isolates obtained from single spore isolation were used
for morphological and molecular assessments.
Morphological identication. In the present study, the fungal isolates obtained were provisionally exam-
ined based on morphological features, specically macroscopic and microscopic characteristics. Colony appear-
ance and pigmentation were observed at the macroscopic level. Under a dissecting microscope, the structure of
the conidiomata was observed and photographed (EZ4, Leica Microsystem, Germany). e microscopic features
such as conidia, conidiogenous cells, and paraphyses were observed using a light microscope (CX41, Olympus,
Japan) and a camera (KY-F55BE, JVC, Japan). e average size of 30 randomized conidia was measured and
recorded. Each fungal isolate was cultured onto carnation leaf agar (CLA) and incubated at 25°C ± 2°C for
7days to observe the structures of conidiomata, conidia, conidiogenous cells, and paraphyses.
Molecular identication and phylogenetic analysis. To corroborate the identity of the fungal iso-
lates of the present study, molecular identication and characterization was carried out. e fungal isolates
were cultured in potato dextrose broth (PDB) and subjected to incubation at 25°C ± 2°C for 5 to 7days. e
mycelia that grew on the surface of PDB were collected, placed on the sterile lter paper (Whatman No. 1),
and le to dry for 10min. Using a sterile mortar and pestle, the dried mycelia were ground to a ne powder in
liquid nitrogen. en, 0.05g of the ne powdered mycelia was placed in a 1.5ml microcentrifuge tube for DNA
extraction. e InnuPREP Plant DNA kit (Analytik Jena, Germany) was used to extract DNA by referring to the
manufacturer’s protocols. For amplication of internal transcribed spacer (ITS), elongation translation factor
1-alpha (tef1-α), β-tubulin (tub2), and RNA polymerase subunit II (rpb2), primer pairs of ITS1 (TCC GTA GGT
GAA CCT GCG G)/ITS4 (TCC TCC GCT TAT TGA TAT GC)69, EF1-688F (CGG TCA CTT GAT CTA CAA GTGC)/
EF1-1251R (CCT CGA ACT CAC CAG TAC CG)30, Bt2a (GGT AAC CAA ATC GGT GCT GCT TTC )/Bt2b (ACC
CTC AGT GTA GTG ACC CTT GGC )70, and rpb2-LasF (GGT AGC GAC GTC ACT CCT )/rpb2-LasR (GCG CAA
ATA CCC AGA ATC AT)52 were adopted, respectively. A reaction mixture of 50µl was prepared by adding 8µl of
green buer (Promega, USA), 8µl of MgCl2 (Promega, USA), 1µl of deoxynucleotide triphosphate polymerase
(dNTP) (Promega, USA), 8µl of each primer (Promega, USA), 0.3µl of Taq polymerase (Promega, USA), 1µl of
genomic DNA, and sterile distilled water to obtain a total volume of 50µl. e following conditions were used
in the polymerase chain reaction (PCR) with the MyCycler™ ermal Cycler (Bio-rad, Hercules, USA): Initial
denaturation at 95°C for 7min (ITS)/5min (tef1-α and tub2)/2min (rpb2), then 25 cycles (ITS)/30 cycles (tef1-α
and tub2)/35 cycles (rpb2) of denaturation at 94°C for 1min (ITS)/30s (tef1-α, tub2, and rpb2), annealing at
50°C for 1min (ITS)/55°C for 45s (tef1-α and tub2)/54°C for 30s (rpb2), extension at 72°C for 1min (ITS
and rpb2)/90s (tef1-α and tub2), and nal extension at 72°C for 10min (ITS, tef1-α, and tub2)/8min (rpb2).
Figure4. Sampling sites of diseased eobroma cacao in several states of Malaysia.
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e PCR products were electrophoresed for 90min at 80V and 400mA in a 1.0% agarose gel (Promega, USA)
containing FloroSafe DNA stain (First Base) in a 1.0× Tris–borate EDTA buer. e Bio-Rad Molecular Imager®
Gel Doc™ XR System and Bio-Rad Quantity One® Soware were used to view and photograph the gel. e size of
the amplied PCR products was determined using a 100bp GeneRulers™ DNA ladder (ermo Scientic, USA).
e PCR products were sent to the First BASE Laboratories Sdn Bhd in Seri Kembangan, Malaysia, for DNA
purication and sequencing.
e sequences obtained were compared, and phylogenetic analysis was performed using the Molecular
Evolutionary Genetic Analysis (MEGA7) soware71. e nucleotide homogeneity of the resulting consensus
sequences was assessed by comparing with other sequence data in the GenBank database using Basic Local
Alignment Search Tools (BLAST) (https:// blas t. nc b i . nlm. nih. gov/ Blast. cgi). All sequences obtained were submit-
ted to the GenBank database. Table2 lists the sequences from the present study and the reference isolates used
for phylogenetic analysis. e phylogenetic classication of the isolates from the present study was performed
by analyzing the combination of multi-sequence alignments of fungal isolates and reference isolates in MEGA7
using the maximum likelihood (ML) method. e ML tree of combined genes was constructed using the Tamura
3-parameter model72 and 1000 bootstrap replicates73.
Pathogenicity tests. A total of 57 fungal isolates were assessed for pathogenicity on leaves (13 isolates),
stems (20 isolates), and pods (24 isolates) of T. cacao using KM clone. e 1-year-old healthy seedlings of T.
cacao grown using clay loam soil with a pH of 6.5–7 in polythene bags; and healthy mature pods (5months old
and 17cm in size) taken from 3-year-old trees were purchased from the MCB. e seedlings were placed in the
plant house of the School of Biological Sciences, Universiti Sains Malaysia (USM) at a temperature of 26°C to
32°C.
A fungal mycelial plug used as an inoculum was prepared from a 7-day-old PDA culture using a sterile cork
borer (5mm diameter). For control, the PDA plugs without fungal mycelia were prepared from the blank PDA
using the same methods. Pathogenicity tests for all fungal isolates were performed twice. e tests were carried
out on 84 healthy attached young leaves (84 seedlings), 126 stems (126 seedlings), and 150 detached pods of T.
cacao. e targeted plant parts were surface-sterilized with 70% ethanol prior to inoculation.
To inoculate 13 fungal isolates on leaves of T. cacao, a total of 84 healthy leaves (78 for the fungal treatment
and six for the control) from 84 seedlings of T. cacao were used for two pathogenicity tests. Each surface-sterilized
leaf was aseptically pricked at one point with a sterile toothpick represented a replicate. For each pathogenicity
test, three replicates were performed for each fungal isolate, using three dierent leaves from three dierent
seedlings. Controls were performed in the same ways but treated with the blank PDA plugs. A sterile scalpel
was used to inoculate control and mycelial plugs onto the control and treatment points, respectively. e plugs
were wrapped in sterile cotton wool and xed to the leaf with cellophane tape to avoid dryness. Each inoculated
leaf was covered in a sterile zip lock bag. e inoculated seedlings were kept in the plant house of the School of
Biological Sciences, USM for 9days at temperatures ranging from 26 to 32°C.
A total of 126 healthy stems of T. cacao (126 seedlings) were used to inoculate 20 fungal isolates for twice
pathogenicity tests. A small wound (0.5cm) was created on the sterilized surface of each stem by removing the
bark with a sterile scalpel. For each pathogenicity test, three wounded stems from three dierent seedlings were
used to inoculate each fungal isolate, representing triplicates. Control was treated similarly using the blank
PDA plugs. Using a sterile scalpel, the mycelial and control plugs were placed on the wounded points, with the
mycelium positioned towards the cambium. e moisture of the plugs was maintained by wrapping in sterilized
cotton and sealing with paralm. All the inoculated seedlings were incubated in the plant house of the School
of Biological Sciences, USM at temperatures ranging from 26 to 32°C.
Twice pathogenicity tests conducted on healthy detached cocoa pods involved 150 pods (144 for the fungal
treatment and six pods for the control). Control and fungal treatments were inoculated on dierent pods to avoid
symptoms overlapping if both were performed on the same pods. For each pathogenicity test, a wound point was
created on the three dierent pods for each fungal isolate by piercing the pod surface with a sterile cork borer.
en, 5mm mycelial plugs with the mycelium facing the surface of the pods were placed on the wounded points.
e three control pods were treated in the same way but using the blank PDA plugs. To retain moisture, all the
plugs were wrapped with sterilized cotton wool and the cotton was xed with cellophane tape. e inoculated
cocoa pods were incubated for 12days at 25°C ± 2°C in sterilized trays and covered with transparent plastic to
maintain humidity.
e area of the lesion developed on the inoculated leaves, stems, and pods of T. cacao was measured using
grid paper adopted by Parker etal.74 with slight modications. e area of diseased lesion was calculated by
multiplying the number of small squares covering the lesion with the value calculated for one small square. Dif-
ferences in lesion area were evaluated using the one-way method ANOVA and means were compared with the
Tukey’s test (p < 0.05) using the soware IBM SPSS Statistics version 26. To conrm Koch’s postulates, fungi from
symptomatic inoculated leaves, stems, and pods of T. cacao were reisolated and reidentied using morphological
characteristics.
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Species Isolate Host Location
GenBank accession number
ReferencesITS tef1-α tub2 rpb2
Lasiodiplodia brasiliensis CBS123095 eobroma cacao Cameroon MT587423 MT592135 MT592615 MT592309 Zhang etal.75
L. brasiliensis CBS115447 Psychotria tutcheri Hong Kong MT587422 MT592134 MT592614 MT592308 Zhang etal. 75
L. brasiliensis CMM4015aMangifera indica Brazil JX464063 JX464049 MT592614 MT592308 Marques etal.76
L. brasiliensis CSM11 eobroma cacao Venezuela MF436018 MF436006 MF435998 MT592308 Mohali-Castillo and
Stewart19
Lasiodiplodia citricola CBS124707aCitrus sp. Iran GU945354 GU945340 KU887505 KU696351 Cruywagen etal.52;
Abdollahzadeh etal.55
L. citricola CBS124706 Citrus sp. Iran GU945353 GU945339 KU887504 KU696350 Cruywagen etal.52;
Abdollahzadeh etal.55
Lasiodiplodia crassispora CBS118741aSantalum album Australia DQ103550 DQ103557 KU887506 KU696353 Cruywagen etal.52
L. crassispora CBS125626 Vitis vinifera South Africa MT587424 DQ103557 MT592617 MT592312 Zhang etal.75
L. crassispora CMW33262 Adansonia sp. Unknown KU887068 DQ103557 KU887426 KU887364 Cruywagen etal.52
Lasiodiplodia euphor-
biicola CMM3609aJatropha curcas Brazil KF234543 KF226689 KF254926 KU887367 Machado etal.77
L. euphorbiicola CMM3651 Jatropha curcas Brazil KF234553 KF226711 KF254937 KU887367 Machado etal.77
L. euphorbiicola CMW33268 Adansonia sp. Unknown KU887131 KU887008 KU887430 KU887367 Cruywagen etal.52
Lasiodiplodia hormoz-
ganensis CBS124709aOlea sp. Iran GU945355 GU945343 KU887515 KU696361 Cruywagen etal.52;
Abdollahzadeh etal.55
L. hormozganensis CBS124708 Mangifera indica Iran GU945356 GU945344 KU887514 KU696360 Cruywagen etal.52;
Abdollahzadeh etal.55
Lasiodiplodia iraniensis CBS124710aSalvadora persica Iran GU945348 GU945336 KU887516 KU696363 Cruywagen etal.52;
Abdollahzadeh etal.55
L. iraniensis CBS124711 Juglans sp. Iran GU945347 GU945335 KU887517 KU696362 Cruywagen etal.52;
Abdollahzadeh etal.55
L. iraniensis CMW35881 Adansonia sp. Unknown KU887092 KU886970 KU887464 KU887388 Cruywagen etal.52
Lasiodiplodia lignicola CBS134112aDead wood ailand JX646797 KU887003 JX646845 KU696364 Cruywagen etal.52; Liu
etal.78
L. lignicola MFLUCC110656 Dead wood ailand JX646798 KU887003 JX646846 KU696364 Cruywagen etal.52; Liu
etal.78
Lasiodiplodia mahajan-
gana CBS124925aTerminalia catappa Madagascar FJ900595 FJ900641 KU887518 KU696365 Cruywagen etal.52;
Begoude etal.79
L. mahajangana CBS124926 Terminalia catappa Madagascar FJ900596 FJ900642 KU887519 KU696366 Cruywagen etal.52;
Begoude etal.79
Lasiodiplodia marga-
ritacea CBS122519aAdansonia gibbosa Australia EU144050 EU144065 KU887520 KU696367 Cruywagen etal.52
L. margaritacea CBS138289 Combretum elae-
agnoides Namibia KP872320 KP872349 KP872379 KP872429 Zhang etal.75
L. margaritacea CBS138290 Combretum collinum Zambia KP872321 KP872350 KP872380 KP872430 Zhang etal.75
Lasiodiplodia mediter-
ranea CBS137783aQuercus ilex Italy KJ638312 KJ638331 KU887521 KU696368 Cruywagen etal.52;
Linaldeddu etal.80
L. mediterranea CBS137784 Vitis vinifera Italy KJ638311 KJ638330 KU887522 KU696369 Cruywagen etal.52;
Linaldeddu etal.80
Lasiodiplodia pseudothe-
obromae CBS116459aGmelina arborea Costa Rica EF622077 EF622057 EU673111 KU696376 Alves etal.30; Phillips
etal.81
L. pseudotheobromae CBS116460 Acacia mangium Costa Rica MT587433 MT592145 KU198428 MT592322 Zhang etal.75
L. pseudotheobromae CBS130991 Mangifera indica Egypt MT587433 MT592145 MT592629 MT592325 Zhang etal.75
L. pseudotheobromae I46 eobroma cacao Puerto Rico MK693211 MK693707 MK693702 KU696376 Serrato-Diaz etal.17
Lasiodiplodia theo-
bromae CBS164.69aFruit on coral reef coast Indonesia: New Guinea AY640255 AY640258 EU673110 KU696383 Cruywagen etal.52
L. theobromae CBS214.50 Cajanus cajan India MT587440 MT592152 MT592637 MT592333 Zhang etal.75
L. theobromae CMW13490 Eucalyptus urophylla Venezuela: Acarigua KY473071 KY473019 KY472962 KY472888 Mehl etal.82
L. theobromae CMM4019 Mangifera indica Brazil JX464096 JX464026 EU673110 KU696383 Marques etal.76
L. theobromae CSM57 eobroma cacao Venezuela MF436029 MF436017 MF435999 KU696383 Mohali-Castillo and
Stewart19
L. theobromae M400 eobroma cacao USA: Puerto Rico MN446021 MN536705 MN536694 KU696383 Puig etal.25
L. theobromae NS2F eobroma cacao Malaysia: Negeri
Sembilan OL831055 OL863319 OL863262 OL863376 is study
L. theobromae M3F eobroma cacao Malaysia: Melaka OL831056 OL863320 OL863263 OL863377 is study
L. theobromae M4F eobroma cacao Malaysia: Melaka OL831057 OL863321 OL863264 OL863378 is study
L. theobromae NS7F eobroma cacao Malaysia: Negeri
Sembilan OL831058 OL863322 OL863265 OL863379 is study
L. theobromae NS8F eobroma cacao Malaysia: Negeri
Sembilan OL831059 OL863323 OL863266 OL863380 is study
Continued
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Species Isolate Host Location
GenBank accession number
ReferencesITS tef1-α tub2 rpb2
L. theobromae PP9F eobroma cacao Malaysia: Pulau Pinang OL831060 OL863324 OL863267 OL863381 is study
L. theobromae PP11F eobroma cacao Malaysia: Pulau Pinang OL831061 OL863325 OL863268 OL863382 is study
L. theobromae J13F eobroma cacao Malaysia: Johor OL831062 OL863326 OL863269 OL863383 is study
L. theobromae J15F eobroma cacao Malaysia: Johor OL831063 OL863327 OL863270 OL863384 is study
L. theobromae J16F eobroma cacao Malaysia: Johor OL831064 OL863328 OL863271 OL863385 is study
L. theobromae M19F eobroma cacao Malaysia: Melaka OL831065 OL863329 OL863272 OL863386 is study
L. theobromae PE20F eobroma cacao Malaysia: Perak OL831066 OL863330 OL863273 OL863387 is study
L. theobromae PE22F eobroma cacao Malaysia: Perak OL831067 OL863331 OL863274 OL863388 is study
L. theobromae PP23F eobroma cacao Malaysia: Pulau Pinang OL831068 OL863332 OL863275 OL863389 is study
L. theobromae K25F eobroma cacao Malaysia: Kedah OL831069 OL863333 OL863276 OL863390 is study
L. theobromae K27F eobroma cacao Malaysia: Kedah OL831070 OL863334 OL863277 OL863391 is study
L. theobromae S30F eobroma cacao Malaysia: Selangor OL831071 OL863335 OL863278 OL863392 is study
L. theobromae PE31F eobroma cacao Malaysia: Perak OL831072 OL863336 OL863279 OL863393 is study
L. theobromae PE32F eobroma cacao Malaysia: Perak OL831073 OL863337 OL863280 OL863394 is study
L. theobromae S34F eobroma cacao Malaysia: Selangor OL831074 OL863338 OL863281 OL863395 is study
L. theobromae S35F eobroma cacao Malaysia: Selangor OL831075 OL863339 OL863282 OL863396 is study
L. theobromae PR36F eobroma cacao Malaysia: Perlis OL831076 OL863340 OL863283 OL863397 is study
L. theobromae PR37F eobroma cacao Malaysia: Perlis OL831077 OL863341 OL863284 OL863398 is study
L. theobromae PE39F eobroma cacao Malaysia: Perak OL831078 OL863342 OL863285 OL863399 is study
L. theobromae K41L eobroma cacao Malaysia: Kedah OL831081 OL863343 OL863286 OL863400 is study
L. theobromae K42L eobroma cacao Malaysia: Kedah OL831082 OL863344 OL863287 OL863401 is study
L. theobromae PR43L eobroma cacao Malaysia: Perlis OL831083 OL863345 OL863288 OL863402 is study
L. theobromae PR44L eobroma cacao Malaysia: Perlis OL831084 OL863346 OL863289 OL863403 is study
L. theobromae PE45L eobroma cacao Malaysia: Perak OL831085 OL863347 OL863290 OL863404 is study
L. theobromae PE46L eobroma cacao Malaysia: Perak OL831086 OL863348 OL863291 OL863405 is study
L. theobromae S47L eobroma cacao Malaysia: Selangor OL831087 OL863349 OL863292 OL863406 is study
L. theobromae S48L eobroma cacao Malaysia: Selangor OL831088 OL863350 OL863293 OL863407 is study
L. theobromae S49L eobroma cacao Malaysia: Selangor OL831089 OL863351 OL863294 OL863408 is study
L. theobromae M50L eobroma cacao Malaysia: Melaka OL831090 OL863352 OL863295 OL863409 is study
L. theobromae M51L eobroma cacao Malaysia: Melaka OL831091 OL863353 OL863296 OL863410 is study
L. theobromae NS52L eobroma cacao Malaysia: Negeri
Sembilan OL831080 OL863354 OL863297 OL863411 is study
L. theobromae NS53L eobroma cacao Malaysia: Negeri
Sembilan OL831079 OL863355 OL863298 OL863412 is study
L. theobromae J54S eobroma cacao Malaysia: Johor OL831092 OL863356 OL863299 OL863413 is study
L. theobromae J55S eobroma cacao Malaysia: Johor OL831093 OL863357 OL863300 OL863414 is study
L. theobromae J56S eobroma cacao Malaysia: Johor OL831094 OL863358 OL863301 OL863415 is study
L. theobromae J57S eobroma cacao Malaysia: Johor OL831095 OL863359 OL863302 OL863416 is study
L. theobromae J58S eobroma cacao Malaysia: Johor OL831096 OL863360 OL863303 OL863417 is study
L. theobromae J59S eobroma cacao Malaysia: Johor OL831097 OL863361 OL863304 OL863418 is study
L. theobromae NS60S eobroma cacao Malaysia: Negeri
Sembilan OL831098 OL863362 OL863305 OL863419 is study
L. theobromae NS61S eobroma cacao Malaysia: Negeri
Sembilan OL831099 OL863363 OL863306 OL863420 is study
L. theobromae NS62S eobroma cacao Malaysia: Negeri
Sembilan OL831100 OL863364 OL863307 OL863421 is study
L. theobromae M63S eobroma cacao Malaysia: Melaka OL831101 OL863365 OL863308 OL863422 is study
L. theobromae M64S eobroma cacao Malaysia: Melaka OL831102 OL863366 OL863309 OL863423 is study
L. theobromae S65S eobroma cacao Malaysia: Selangor OL831103 OL863367 OL863310 OL863424 is study
L. theobromae S66S eobroma cacao Malaysia: Selangor OL831104 OL863368 OL863311 OL863425 is study
L. theobromae PE67S eobroma cacao Malaysia: Perak OL831105 OL863369 OL863312 OL863426 is study
L. theobromae PE68S eobroma cacao Malaysia: Perak OL831106 OL863370 OL863313 OL863427 is study
L. theobromae PP69S eobroma cacao Malaysia: Pulau Pinang OL831107 OL863371 OL863314 OL863428 is study
L. theobromae PP70S eobroma cacao Malaysia: Pulau Pinang OL831108 OL863372 OL863315 OL863429 is study
L. theobromae PP71S eobroma cacao Malaysia: Pulau Pinang OL831109 OL863373 OL863316 OL863430 is study
L. theobromae J72S eobroma cacao Malaysia: Johor OL831110 OL863374 OL863317 OL863431 is study
L. theobromae J73S eobroma cacao Malaysia: Johor OL831111 OL863375 OL863318 OL863432 is study
Continued
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Data availability
All sequence data are available in NCBI GenBank [https:// www. ncbi. nlm. nih. gov/ genba nk/] following the
accession numbers [OL831055–OL831111 (ITS); OL863319–OL863375 (tef1-α); OL863262–OL863318 (tub2);
OL863376–OL863432 (rpb2)] in the manuscript. All data analyzed during this study are included in this pub-
lished article and its supplementary information les.
Received: 18 February 2022; Accepted: 19 May 2022
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L. viticola CBS128314 Chardonel USA HQ288228 HQ288270 HQ288307 KU696386 Cruywagen etal.52
Botryosphearia dothidea CBS115476 Prunus sp. Switzerland KF766151 AY236898 MT592470 DQ677944 Slippers etal.83
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Acknowledgements
e authors thank the Malaysian Cocoa Board (MCB) for permission to collect samples and provide healthy
seedlings for pathogenicity tests. Special thanks to the MCB sta who have assisted in the eldwork activities.
Author contributions
A.R.H-S.: conceptualization, methodology, formal analysis, investigation, writing-original dra preparation.
N.M.I.M.N., L.Z., Y.-H.L.: writing-review and editing. M.H.M.: conceptualization, methodology, investigation,
writing-review and editing, supervision.
Funding
is research was funded by Fundamental Research Grant Scheme (FRGS/1/2019/WAB01/USM/02/1) from
Ministry of Higher Education, Malaysia.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 13057-9.
Correspondence and requests for materials should be addressed to M.H.M.
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