RESEARCH ARTICLE

Identification of cassava black stem and root rot agents

in Thailand
Rungthip Sangpueak1, Suttisa Duchanee2, Chanon Saengchan1, Narendra Kumar Papathoti1,
Nguyen Huy Hoang1, Toan Le Thanh3, Piyaporn Phansak4, and Natthiya Buensanteai1*
1
Suranaree University of Technology, School of Crop Production Technology, Institute of Agricultural Technology,
30000, Nakhon Ratchasima, Thailand.
2
National Science and Technology Development Agency (NSTDA), 12120, Pathum Thani, Thailand.
3
Can Tho University, College of Agriculture, Department of Plant Protection, 900000, Can Tho, Viet Nam.
4
Nakhon Phanom University, Faculty of Science, Division of Biology, 48000, Nakhon Phanom, Thailand.
*
Corresponding author (natthiya@sut.ac.th).

Received: 26 May 2022; Accepted: 8 August 2022; doi:

ABSTRACT
Cassava (Manihot esculenta Crantz) black stem and root rot (BSRR) caused by complex fungi is one of the
most serious fungal disease of cassava in Thailand. The objective of this study was to identify the causal agent
of BSRR disease of cassava in Nakhon Ratchasima province, Thailand. From June 2015 to May 2018, the
outbreak areas in three districts of Nakhon Ratchasima province were surveyed for cassava plants with the
characteristic symptoms. From 1800 diseased samples, 139 fungal pathogens were isolated and separated by
morphological traits. Among them, 33 fungal isolates were subsequently tested for pathogenicity on detached
stems and roots of CMR 43-08-89, a susceptible cassava cultivar, and proved to be pathogenic with different
levels of aggressiveness. Next, PCR amplification of DNA of eight pathogenic isolates with high
aggressiveness using two sets of universal primers ITS1/ITS4 and Ef1-688F/Ef1-1251R, revealed that the
TEF1-α gene region could be used for the identification and classification at the species level. The analysis
results fit well with that of the morphological studies on growth and colonial characteristics of the eight
isolates. Based on the GenBank database, they were identified as Lasiodiplodia theobromae, L.
euphorbiaceicola and Neoscytalidium dimidiatum.

Key words: Cassava disease, Lasiodiplodia spp., Manihot esculenta, Neoscytalidium dimidiatum.

INTRODUCTION
Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. It is categorized as an
agro-based industrial crop with well-growing industries and markets in and outside the country. Thailand has
been the world-leading exporter of cassava products with a production volume of 31 million tons in 2018-2019
(Arunmas, 2019). Unlike in Africa or South America, in which cassava product is mainly consumed as a staple
food; in Thailand, it has been mainly used as raw materials in industries for the production of tapioca starch,
modified starch, ethanol, and animal feed. As such, it has been treated as a cash crop benefiting millions of
subsistence Thai farmers. Cassava has changed its roles to a profitable crop on account of the classification of
many process industries in which cassava can be used as industrial basic materials. To encourage food and
energy industries, an escalation of cassava production by using good agricultural practices has been promoted
in Thailand (Sangpueak et al., 2018). Since then, its cultivation has drastically changed in terms of planting
acreage and practices to meet the continuously increasing demand. As a result, the outbreak and severity of
pests and diseases have increased since 2010 (Sangpueak et al., 2018).
Cassava plants could be attacked by more than 34 kinds of pathogens, causing various degrees of losses
(Sangpueak et al., 2018; Maruthi et al., 2019). In South America, Africa, and India, black stem and root
rot (BSRR) has been considered one of the important diseases on the account that it can cause as much as

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80%-100% loss to the cassava crops. The fungi Lasiodiplodia theobromae and Neoscytalidium dimidiatum
have been identified to be the causal agents of the disease (Nyaka et al., 2015; Boas et al., 2017; Bodah, 2017).
In addition, there have been reports in Brazil and neighboring countries that it was caused by the L.
euphorbiaceicola, L. pseudotheobromae, L. hormozganensis, L. parva, L. brasiliensis, L. caatinguensis, L.
iranensis, L. laeliocattleyae, N. hyalina and Macrophomina pseudophaseolina (Machado et al., 2014a; Brito et
al., 2019; 2020). The disease is widespread during the hot and wet seasons. Usually, the diseased plant bark
becomes darkened and has a split that extends upward on the stem. Moreover, Botryosphaeriaceae species are
complex with various economic crops hosts, including citrus, coconut, cactus prickly pear, coconut palms, tea
(Phillips et al., 2013; Rosado et al., 2016; Feijo et al., 2019; Santos et al., 2020; Rathnayaka et al., 2021).
Although L. theobromae and N. dimidiatum are the two fungi commonly reported as the causal agents, their
identification has been mainly based on their morphology, which could be inadequate to characterize fungus at
a species level. Presently, studying its morphological and molecular characteristics together has been generally
accepted as a standard method for the identification of species and taxonomy of pathogens (Machado et al.,
2014a; 2014b). The internal transcribed spacer (ITS) region has been used as a standard barcode for detecting
many fungal pathogens (Schoch et al., 2012). However, the imprecision of some species could not be
distinguished when only the ITS region was used (Ismail et al., 2012; Marques et al., 2013). Therefore, some
additional sequences of the translation elongation factor EF-1 alpha (TEF1-) gene have to be used along with
the ITS for confirmation of the Botryosphaeriaceae species (Marques et al., 2013; Rosado et al., 2016; Brito et
al., 2020; Santos et al., 2020). Even though BSRR disease was found in Thailand many years ago, its causal
agent has not been precisely elucidated. In this study, identification of the causal fungi of BSRR was performed
using a combination of morphological and molecular studies.

MATERIALS AND METHODS

Diseased sample collection and isolation of the pathogens
Field surveys were carried out from June 2015 to May 2018 in cassava (Manihot esculenta Crantz) plantations
of Nakhon Ratchasima province, Thailand, to collect 1800 black stem and root rot (BSRR) diseased samples
with collar rot, stem rot, leaf fall, wilt, and root rot symptoms. Typical symptoms of BSRR disease were
vascular discoloration, offensive odor, blackening, decaying of the stem, and rotting of roots. Small-sized black
pycnidia and dieback symptoms were also observed on the diseased cassava stems and stalks.
To isolate the causal pathogen, the diseased samples of cassava were cleaned by washing with running tap
water. Subsequently, the diseased portions were cut into small pieces, about 2 mm in size and surface-sterilized
with 1% sodium hypochlorite (NaOCl) for 2-3 min. They were further washed with sterile distilled water at
least three times before being dried between sterilized filter papers. The cassava samples were then placed on
water agar (WA) medium and incubated at room temperature for 48 h. After that, the growing hyphal tips were
transferred onto potato dextrose agar (PDA).
To induce spore formation, fresh cultures were transferred onto sterilized toothpicks soaked in potato
dextrose broth (PDB) in Petri dishes and incubated at 25 °C with a 12:12 h light/dark cycle for 3 wk.
Subsequently, single spore-isolates were taken from each of the pycnidium formed on the toothpicks. The pure
isolates were cultured on PDA and stored at 4 °C for further experiments (modified Phillips et al., 2013;
Machado et al., 2014a; 2014b).

Morphological characterization
Each pure fungal isolate was transferred onto PDA, and a double-sterilized toothpick was placed adjacent to it,
after that the agar plate was incubated at 25 °C with a regime of 12:12 h photoperiod for up to 3 wk to stimulate
the formation of pycnidia. All relevant morphological characters of the isolates were studied using a light
microscope (slightly modified from Machado et al., 2014a; 2014b).

Pathogenicity tests
Each fungal isolate was grown on PDA (7 d, room temperature) and used for inoculation of pathogenicity test.
Stem inoculation. The stem inoculation method was adapted from Trakunyingcharoen et al. (2013) and
Chen et al. (2016). Stalks of healthy cassava plants, susceptible variety (CMR 43-08-89), were cut into 10 cm
portions and surface-sterilized with 10% NaOCl for 1 min. Next, the portions of cassava stalk were washed out
with sterilized water three times and blotted dried with sterile paper towel. To inoculate the fungus, a 6-mm-

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diameter hole was cut into the bark using a sterile cork borer. Then, a piece of PDA slice with 7-d-old fungal
culture was inserted into the hole and sealed with a strip of parafilm. The inoculated cassava portions were
finally kept in a moist chamber for observation. After 3 wk, the parafilm was removed from the bark and the
fungus was re-isolated and rechecked (Trakunyingcharoen et al., 2013; 2015).
Root inoculation. The inoculation was done similarly to that of the stem inoculation but the storage root
portion was used instead. The inoculated roots were kept in moist chambers for 1 wk for observation (Ismail et
al., 2012; Li et al., 2015). The pathogens from infection lesions were re-isolated in culture media and
rechecked.
Disease severity scores of each isolate were evaluated during the observation using the scale described by
Trakunyingcharoen et al. (2013) and Chen et al. (2016).

Fungal DNA extraction, sequencing, and phylogenetic studies

One gram of pathogenic mycelia was frozen and ground in liquid nitrogen, then transferred into a 1.5 mL
microcentrifuge tube, then 500 μL extraction buffer (0.2 M Tris-HCl, 0.25 M NaCl, 25 mM EDTA, and 2%
sodium dodecyl sulfate, pH 8.5) was added. The tube was incubated at 65 °C for 30 min and then centrifuged at
14000 rpm, for 10 min. The supernatant was transferred into a new tube, and 200 μL chloroform:isoamyl
alcohol (24:1 v/v) was added. The mixture was vortexed for 30 s and then centrifuged at 14000 rpm for 10 min.
Subsequently, the supernatant was transferred to a new tube, and 2 volumes of ice-cold absolute ethanol were
added. The mixture was carefully mixed by vortexing for 30 s, centrifuged at 10000 rpm for 5 min, then, the
supernatant was discarded, and the DNA pellet was washed with 70% ethanol (400 μL). The washed pellet was
dried under a vacuum condition for 30 min and resuspended in 50 μL TE buffer (0.1 mM EDTA and 10 mM
Tris-HCl, pH 8.5) by gentle agitation to dissolve the DNA and stored frozen (-20 °C) until further use
(Sangpueak et al., 2018).
The PCR amplification of the fungal DNA was done using primers ITS1 (5’-TCC GTA GGT GAA CCT
GCG G-3’)/ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) (Fernández-Herrera et al., 2017), and Ef1-688F
(5'-CGG TCA CTT GAT CTA CAA GTG C-3')/Ef1-1251R (5'-CCT CGA ACT CAC CAG TAC CG-3')
(Wang and Song, 2021). The 25 μL reaction mix was prepared as follow: Taq DNA polymerase (0.2 μL,
Invitrogen by Gibthai Company, Thailand), 5 μL 10X buffer, 0.5 μL 50 mM MgCl2, 0.75 μL 10 mM dNTP, 0.5
μL 10 μM primers, 1 μL genomic DNA and adjusted to the final volume with sterile deionized water. The PCR
amplification was carried out using a thermal cycler (PCR MJ Mini, BIO-RAD, Hercules, California, USA).
Following an initial denaturation at 95 °C for 1 min, the DNA templates were amplified for 35 cycles. Each
cycle consisted of a denaturation step at 95 °C for 1 min, an annealing step at 55 °C for 1 min (for TEF1-) or
52 °C for 1 min (for ITS), and an extension step at 72 °C for 2 min. A final extension step (72 °C for 10 min)
was included at the end of the cycle. Amplification products were separated on 1% (w/v) agarose gels in Tris-
acetate-EDTA (TAE) buffer. Before electrophoresis, gels were stained with 6X DNA loading buffer (Novel
Juice, GeneDireX, Taiwan, China), and the DNA bands were visualized with UV light (UVP GelDoc-It2
Imager, Analytik Jena, Jena, Canada). The instrument was also used for data and image capturing (Sompong et
al., 2012; Sangpueak et al., 2018).
Purified PCR products were twice sequenced by the Pacific Scientific (Jakarta Barat, Indonesia), and
analyzed using the Chromas Lite software (Technelysium Pty Ltd., South Brisbane, Queensland, Australia) to
determine the consensus sequence. Nucleotide sequence alignment was performed with the BLAST-N program
and algorithm MEGABLAST, based on NCBI gene bank data (National Center for Biotechnology Information,
Bethesda, Maryland, USA; http://www.ncbi.nlm.nih.gov) (Phillips et al., 2013).

Table 1. Black stem and root rot disease incidence in cassava plants surveyed in Nakhon Ratchasima
province, Thailand during June 2015 to May 2018.
Surveyed cassava Inspected cassava
Location fields samples Disease incidence
Nr Nr %
Khon Buri 6 600 34
Soeng Sang 9 900 46
Nong Bun Mak 3 300 28
Total 18 1800 -

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 The nucleotide sequences of Lasiodiplodia and Neoscytalidium obtained from the Genbank database were
analyzed using the Clustal W program to arrange relationships (multiple sequence aliment) for neighbor-
joining (NJ) Unweighted pairs group methods with arithmetic mean (UPGMA) and Maximum likelihood (ML)
by MEGA 6 (Tamura et al., 2013; De Silva et al., 2019). Bootstrap values were obtained from 1000 NJ
bootstrap replicates. Maximum-parsimony analysis was performed using the heuristic search option with 1000
random taxon additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm to prove
the best and most accurate. Then analyzed together with the morphology and biomolecular characterization and
phylogenetic analysis of pathogens to confirm that cause root rot of cassava.

RESULTS
Sample collection and fungal isolation
A total of 18 cassava fields in three districts of Nakhon Ratchasima province had been surveyed, including
Soeng Sang, Khon Buri, and Nong Bun Mak. From 1800 cassava samples inspected, disease incidences among
districts were varied, with Soeng Sang being the highest at 46%, while those of Khon Buri and Nong Bun Mak
were at 34% and 28%, respectively (Table 1). The BSRR symptoms could be found on all three parts of the
cassava plants; but most frequently on the stems and roots, starting from 1-mo old plants until harvest. On the
stems, the wounds were initially light brown and turned to black necrotic lesions with numerous small-sized
black pycnidia. The infected stems normally withered or die-back, resulting in plant death. Similar wounds
were also observed on the stalks. The withering or dying plants, when uprooted, showed rotten roots with an
offensive odor and blackening of the woody part. Both affected roots and stems displayed internal discoloration
and dark-browning of vascular regions when cut open (Figure 1).

Morphological studies
In total, 139 isolates of fungal pathogens were obtained from the cassava samples collected in three districts of
Nakhon Ratchasima. Among them, 33 isolates were selected from morphological identification and locations,
50 single-spore representatives were chosen for this study. From their growth habits and morphological
characters, the isolates were identified as Lasiodiplodia and Neoscytalidium in the Botryosphaeriaceae family.
Morphological characterizations of the isolates are as follows:

Figure 1. Symptoms of black stem and root in cassava found during the surveys: Wilted plants (A-
C), root rot (D-G), black pycnidia protruding from the affected bark (H-I), and black stem rot (J).

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 Figure 2. Colonies and conidia of Lasiodiplodia spp.: Colonies on toothpick culture (A, B),
conidiogenous cells (C), paraphyses (D), non-septate immature conidia (E). Scale bar = 10 µm.

Figure 3. Colonies and conidia of Neoscytalidium sp.: Colonies on toothpick culture (A, B), three
celled conidia (C, D), chained arthrospores (E, F). Scale bar = 5 µm.

Group 1, Lasiodiplodia spp.: Twenty-eight isolates were classified into this group. Their colonies were dark
grayish, green-olive to greyish black on PDA medium. Immature conidia were clear, oval-shaped, and one-
celled. Mature conidia were striated, dark brown, oval-shaped and one septum or aseptate (Figure 2). Based on
conidia size, the 28 isolates were subdivided into three subgroups; large (L), medium (M) and small (S). The L
group consisted of three isolates L1STRM, L5KBSH1, and L28SBB, having average conidial sizes at 19.50-
32.80 × 12.40-19.70 μm. The M group, contained 13 isolates; L4STR1, L7STRW, L9STR2, L12SHRD,

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L14LSR, L16BLSR, L18KBSH3, L19KBSH2, L21SRE, L23STWH, L24HRWH, L25SRC, and L26HRWB)
having 19.00-30.10 × 10.10-19.32 μm conidial sizes. The S group contained 12 isolates, namely L2SHSR1,
L3BSR, L6HRD, L8HDBB, L10BBLB, L11HSR2, L13SRTF, L15HDBB, L17STRB, L22HRW, L20FHTB1
and L27SRBT having 12.50-26.30 × 9.51-15.00 μm conidial sizes (Table 2).
Group 2, Neoscytalidium sp.: five isolates including N1SRTTC, N2SHCA, N3DTD2, N4BBPM and
N5STRST were classified into this group. Their colonies were black, growing rapidly on PDA medium. The
conidia were dark brown, smooth, thick-walled, 1-2 and occasionally 3 celled and 10.15-16.25 × 3.63-6.25 µm
in sizes. Similar arthroconidia were also found formed in chains (Figure 3) and averaged 6.14-14.36 × 2.96-
5.12 µm in size (Table 3). Thirty-three isolates representing 139 specimens of the fungal pathogen were chosen
to do the pathogenicity test.

Pathogenicity test
By inoculating the 33 isolates to detached stems and roots of CMR 43-08-89, all of them could infect the plants
but showed different degrees of aggressiveness (Table 4, Figures 4A-4C and Figures 5A-5C).

Table 2. Conidial size, paraphyses size and septation of Lasiodiplodia groups isolated from cassava
plants with black stem and root rot disease in Nakhon Ratchasima, Thailand. 1Average of 50 conidia
for each isolate on water agar medium.
Paraphyses
Species/Isolate Conidial size1 Paraphyses size septation
µm µm
L group
L1STRM 22.87-30.00 × 13.00-19.35 - -
L5KBSH1 19.50-32.80 × 12.40-19.70 - -
L28SBB 22.12-30.00 × 12.50-19.35 - -
M group
L4STR1 20.00-26.30 × 10.10-17.20 60 × 3-4 Aseptate
L7STRW 22.12-30.10 × 12.50-16.52 - -
L9STR2 20.00-26.30 × 10.10-17.20 - -
L12SHRD 20.00-24.00 × 12.00-14.00 74 × 3-4 Aseptate
L14LSR 20.10-24.50 × 10.10-14.00 - -
L16BLSR 24.00-28.00 × 10.36-13.24 - -
L18KBSH3 24.00-28.00 × 10.10-17.20 50 × 3-4 Aseptate
L19KBSH2 19.00-23.50 × 11.00-12.50 - -
L21SRE 24.00-28.00 × 10.10-17.20 - -
L23STWH 20.00-26.30 × 10.10-17.20 - -
L24HRWH 20.00-26.30 × 10.10-13.24 - -
L25SRC 24.00-28.00 × 10.30-12.60 - -
L26HRWB 24.00-28.00 × 12.50-19.32 - -
S group
L6HRD 17.00-23.00 × 10.00-13.00 60 × 3-4 Aseptate
L8HDBB 17.54-23.35 × 10.36-14.45 56 × 3-4 Aseptate
L11HSR2 12.50-22.50 × 10.00-15.00 46 × 3-4 Aseptate
L13SRTF 16.00-23.00 × 9.50-12.50 59 × 3-4 Aseptate
L20FHTB1 17.50-24.10 × 10.10-12.50 56 × 3-4 Aseptate
L27SRBT 20.00-26.00 × 10.00-13.00 63 × 3-4 Aseptate
L2SHSR1 17.54-23.00 × 10.36-13.24 - -
L3BSR 16.50-20.50 × 10.30-12.60 - -
L10BBLB 16.50-22.10 × 10.30-14.50 - -
L15HDBB 19.80-22.00 × 12.10-14.00 - -
L17STRB 17.50-24.10 × 10.10-12.50 - -
L22HRW 18.20-21.50 × 10.00-14.50 - -

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 Table 3. Conidial and arthroconidia sizes of Neoscytalidium spp. isolated from cassava plants with
black stem and root rot disease in Nakhon Ratchasima, Thailand. 1Average of 50 conidia for each
isolate on water agar medium.
Isolate Conidial size1 Arthroconidia size
µm µm
N1SRTTC 11.25-16.25 × 5.00-6.25 8.65-14.36 × 2.96-5.12
N2SHCA 11.25-16.25 × 3.63-4.88 6.25-13.50 × 2.96-5.12
N3DTD2 10.00-15.00 × 4.00-5.00 6.00-14.00 × 3.00-5.00
N4BBPM 10.25-16.25 × 3.63-6.25 6.25-12.36 × 2.96-5.12
N5STRST 11.25-16.25 × 3.63-6.25 7.50-12.36 × 2.96-5.12

Figure 4. Symptoms developed on cassava roots at 7 d after being inoculated with L11HSR2 (A),
L6HRD (B), L18KBSH3 (C), and uninoculated control (D).

Figure 5. Symptoms developed on cassava stems at 15 d after being inoculated with L11HSR2 (A),
L6HRD (B), L18KBSH3 (C), and uninoculated control (D).

Root inoculation: All inoculated roots showed symptoms of fungal infection occurring externally on the
root bark after one week (Figures 4A-4C). Observed symptoms included necrotic bark lesions around the
inoculation points and the root bark was enveloped by a brown to black or greyish mycelia. Such symptoms
were not observed on the control of uninoculated roots (Figure 4D).

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 Stem inoculation: Forty-five days after the inoculation, all isolates could infect the stems exhibiting internal
discoloration and developing large lesions. Some parts of the stems turned into brown to black tissues (Figures
5A-5C). No disease symptoms were observed in the control of uninoculated stems (Figure 5D). Based on the
results of the pathogenicity test, eight representative isolates were chosen to do on the following studies.

Table 4. Pathogenicity and aggressiveness test of 33 isolates on cassava CMR 43-08-89. 1Standard
error ± S.E. different characters mean statistical difference at a probability level of 0.05 by
comparing Duncan’s new multiple range test (DMRT) mean. 2Disease scores 1-5 as follows: 1 =
non-symptomatic, 2 = symptom less than 25% of root/stem, 3 = symptom 25%-50% of root/stem, 4
= symptom 50%-75% of root/stem, 5 = symptom more than 75% of root/stem. 3**Significantly
different at the 0.01 level; ns: nonsignificant.
Root inoculation Stem inoculation
Pathogens isolates Wound size1 Disease scores2 Wound size1 Disease scores2
mm mm
L1STRM 27.50 ± 0.90d-g 3 24.00 ± 2.27c-g 2
L2SHSR1 28.00 ± 1.06c-g 3 29.00 ± 6.75b-e 2
L3BSR 29.25 ± 3.13c-g 3 37.60 ± 1.92ab 3
L4STR1 23.75 ± 2.72e-h 3 34.50 ± 3.75ab 3
L5KBSH1 21.13 ± 2.50f-h 3 11.00 ± 2.35h-j 2
L6HRD 34.25 ± 2.81b-e 4 15.63 ± 1.57g-j 2
L7STRW 23.88 ± 0.67e-h 3 14.38 ± 2.19g-j 2
L8HDBB 30.38 ± 3.01b-f 3 20.00 ± 5.40e-i 2
L9STR2 27.00 ± 2.62d-g 3 30.25 ± 4.27a-e 2
L10BBLB 26.25 ± 1.29d-h 3 22.75 ± 1.31d-g 2
L11HSR2 46.63 ± 2.09a 5 41.25 ± 1.25a 3
L12SHRD 40.08 ± 4.04a-c 4 37.50 ± 6.61ab 3
L13SRTF 35.25 ± 4.75a-e 4 27.75 ± 1.25b-e 2
L14LSR 17.00 ± 2.47g-i 1 33.50 ± 2.90a-d 3
L15HDBB 17.50 ± 1.25g-i 1 30.75 ± 8.10a-e 2
L16BLSR 26.25 ± 4.80d-h 3 8.75 ± 2.14j 2
L17STRB 35.00 ± 4.68a-e 4 16.75 ± 1.18f-j 2
L18KBSH3 26.25 ± 4.80d-h 3 9.50 ± 2.02ij 2
L19KBSH2 35.00 ± 4.68a-e 4 10.50 ± 1.66h-j 2
L20FHTB1 42.38 ± 1.32ab 4 15.00 ± 2.04g-j 2
L21SRE 31.60 ± 3.93b-f 3 33.75 ± 3.75a-d 3
L22HRW 38.13 ± 1.04a-d 4 20.50 ± 1.55e-i 2
L23STWH 13.88 ± 1.94hi 2 16.25 ± 0.52f-j 2
L24HRWH 28.50 ± 6.23c-g 3 0.63 ± 2.13e-h 2
L25SRC 31.50 ± 2.02b-f 3 8.75 ± 0.52j 2
L26HRWB 22.50 ± 6.73e-h 3 7.00 ± 0.46j 2
L27SRBT 40.25 ± 5.09a-c 4 33.75 ± 3.15a-d 3
L28SBB 33.50 ± 2.02b-f 4 10.63 ± 1.49h-j 2
N1SRTTC 26.50 ± 1.95d-g 3 36.00 ± 4.20ab 3
N2SHCA 26.30 ± 1.67d-h 3 26.50 ± 3.30b-f 2
N3DTD2 22.75 ± 0.41e-h 3 36.00 ± 4.85ab 3
N4BBPM 25.50 ± 1.89d-h 3 36.75 ± 2.69ab 3
N5STRST 24.15 ± 0.31e-h 3 30.00 ± 3.11b-e 2
Control 6.50 ± 0.56i 1 6.03 ± 0.02j 1
F-test3 ** **
CV, % 25.87 28.33

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 Figure 6. PCR products of the extracted DNA from fungi causing black stem and root rot of cassava
in Nakhon Ratchasima amplified with ITS1/ITS4 primers (A), Ef1-688F/Ef1-1251R primers (B).
Lane M: Marker 1 kb DNA ladder; lane 1: L6HRD; lane 2: L11HSR2; lane 3: L12SHRD lane 4:
L13SRTF; lane 5: L18KBSH; lane 6: L20FHTB1; lane 7: L22HRW; lane 8: L27SRBT; lane 9:
N1SRTTC; lane 10: N3DTD2.

Table 5. Similarity of DNA sequences of the Lasiodiplodia and Neoscytalidium isolates causing
black stem and root rot disease in Nakhon Ratchasima (NR), Thailand amplified by ITS primers,
compared to those of the references in the GenBank.
Morphology Reference Reference Sequence
NR isolate nr identifications GenBank species similarity
Accession Nr %
L6HRD Lasiodiplodia sp. MK530047 L. theobromae 96
L11HSR2 Lasiodiplodia sp. KJ596529 L. theobromae 97
L12HSRD Lasiodiplodia sp. KJ596529 L. theobromae 97
L13SRTF Lasiodiplodia sp. MT075444 L. theobromae 95
L18KBSH3 Lasiodiplodia sp. MT497430 L. theobromae 96
L20FHTB1 Lasiodiplodia sp. MT472040 L. theobromae 99
L27SRBT Lasiodiplodia sp. LC275062 L. theobromae 99
N3DTD2 Neoscytalidium sp. MT075446 L. theobromae 96

Table 6. Similarity of DNA sequences of the Lasiodiplodia and Neoscytalidium isolates causing
black stem and root rot disease in Nakhon Ratchasima (NR), Thailand amplified by TEF1-α primers,
compared to those of the references in the GenBank.
Morphology Reference Reference Sequence
NR isolate nr identifications GenBank species similarity
Accession nr %
L6HRD Lasiodiplodia sp. KJ417861 L. euphorbiaceicola 96
L11HSR2 Lasiodiplodia sp. MK495418 L. euphorbiaceicola 96
L12HSRD Lasiodiplodia sp. KJ417861 L. euphorbiaceicola 97
L13SRTF Lasiodiplodia sp. MK495416 L. euphorbiaceicola 96
L18KBSH3 Lasiodiplodia sp. KU507444 L. theobromae 97
L20FHTB1 Lasiodiplodia sp. JX464021 L. theobromae 96
L27SRBT Lasiodiplodia sp. MK495396 L. euphorbiaceicola 95
N3DTD2 Neoscytalidium sp. MK495384 N. dimidiatum 95

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PCR amplification and DNA sequencing
When extracted DNA of eight representative isolates of the stem and root rot fungi (L6HRD, L11HSR2,
L12HSRD, L13SRTF, L18KBSH3, L20FHTB1, L27SRBT and N3DTD2) were amplified using the
combination of ITS1/ITS4 primers, 550 bp fragments of the ITS region were obtained from all of them (Figure
6A). But when the combination of Ef1-688F/Ef1-1251R primers was used, 500 bp fragments of the TEF1-α
region were obtained instead (Figure 6B). After the 550 bp fragments (ITS) had been subsequently sequenced
and further analyzed, the results showed that all of them had 95%-99% similarity to that of L. theobromae in
the GenBank (Table 5).

Figure 7. A neighbor-joining tree derived from the combined sequences of TEF1-α of

Botryosphaeriaceae represent bootstrap values (%) from 1000 replicates. A phylogenetic tree was
conducted using MEGA 6.0 with kimura-two parameter model.

Similar sequencing and analysis had also been performed on the 500 bp fragments (TEF1-α) of the eight
isolates and it was found that those of L18KBSH (accession numbers MT472034) and L20FHTB1 (accession
numbers MT472039) showed 96-97% similarity to that of L. theobromae accession numbers KU507444 and
JX464021 in the GenBank, respectively. Sequences of the five isolates including L6HRD (accession numbers

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MT472028), L11HSR2 (accession numbers MT472030), L12SHRD (accession numbers MT472032),
L13SRTF (accession numbers MT472030), and L27SRBT (accession numbers MT472040) had 95%-96%
likeness to that of L. euphorbiaceicola, while that of N3DTD2 (accession numbers MT472042) shared 95%
similarity to that of N. dimidiatum accession number MK495384 in the GenBank (Tables 5 and 6).
Phylogenetic analysis using the TEF1-α align sequence data set of the eight isolates delineated them into
three distinct species of Botryosphaeriaceae: two species of Lasiodiplodia and one species of Neoscytalidium.
The three species of L. euphorbiaceicola, L. theobromae, and N. dimidiatum were grouped in separate clades
(Figure 7).

DISCUSSION
Results of our research demonstrate that the main causal agents of the BSRR disease of cassava in Thailand are
fungi in the Botryosphaeriaceae family, but using their morphological characteristics alone cannot elucidate
their identity at the species level. The colony and conidial characteristics of the 33 isolates indicated that they
belong to either Lasiodiplodia or Neoscytalidium genus but their species determination based on such
characters is still doubtful. This assumption can be seen when the conidial size was used for the identification
that the Lasiodiplodia having large conidial sizes (L group 19.50-32.80 × 12.40-19.70 μm) could be identified
as L. theobromae (21-31 × 13-15.5 μm), or L. citricola (22-27 × 12-17 μm) or L. crassispora (27-30 × 14-17
μm) (Marques et al., 2013). Similar uncertainty is also applied to those with small-sized conidia (S group
12.50-26.30 × 9.51-15.00 μm) which could be identified as L. euphorbiaceicola (15-23 × 9-12 μm) or L. pava
(16-23.5 × 10.5-13 μm) (Machado et al., 2014a; Brito et al., 2020). Therefore, additional criteria are needed for
the precise identification of these pathogens. Comparing the ITS sequences of the representative fungal isolates
with those of GenBank, all eight isolates were identified as L. theobromae, including N3DTD2 that had been
identified as Neoscytalidium based on ITS unique production of arthroconidia and conidial characters. This
result indicates that the additional criterion of ITS homology that has been used by other researchers is
inadequate for the identification of fungi in this group (Munirah et al., 2017). However, when the homology of
the TEF1-α region was used for the comparison, the eight representative isolates could be distinctively
separated into three species: L. theobromae, L. euphorbiaceicola, and N. dimidiatum. This study has been the
first report of finding N. dimidiatum and L. euphorbiaceicola infecting cassava in Thailand.
Our findings show that the TEF1-α sequence is a more reliable criterion for the identification of
Lasiodiplodia species, is consistent with the study of Berraf-Tebbal et al. (2020). These authors reported that
the TEF1-α sequences are better than the ITS for the determination and phylogenetic analysis of the
Lasiodiplodia species. In the study of Rosado et al. (2016), TEF-1-α was major mostly used to generate and
analyze Lasiodiplodia species. Combination of ITS and β-tubulin was just used to support phylogenetic TEF1-
α. For the disease symptomatology, we found that the symptoms could be found in all parts of cassava plants
but most frequently on the stem and root. In terms of the disease etiology, the finding of this research is similar
to that of De Silva et al. (2019). These authors reported that the major pathogens of cassava root rot disease in
sub-Saharan Africa were identified as L. theobromae, Nattrassia mangiferae (synonyms: Scytalidium hyalinum
or Neoscytalidium hyalinum/N. dimidiatum) and Fusarium spp. Additional major causal agents found in their
study were L. theobromae, Macrophomina phaseolina, Fusarium spp. and Pythium sp. Some of other results
showed that only L. theobromae was extremely aggressive when inoculated into cassava stem cuttings (Vilas
Boas et al., 2017). In addition, Motokura et al. (2014) identified the causal agent of stem rot as L. parva in
Japan. Moreover, Machado et al. (2014b) found that the causal agents of cassava black root rot in Brazil are L.
euphorbiaceicola, L. pseudotheobromae, and N. dimidiatum. In Thailand, Buensanteai and Athinuwat (2012)
initially reported the causal agent of severe stem rot as L. theobromae.

CONCLUSIONS
From the results of this research, it can be concluded that the black stem and root rot (BSRR) disease in
Thailand is caused by more than one fungal species. Lasiodiplodia theobromae, Neoscytalidium dimidiatum,
and L. euphorbiaceicola have been proved to cause symptoms similar to those found in the field. At present,
there is not yet a recommendation for the controlling of this disease in Thailand. Therefore, the knowledge
generated by this research should be of assistance for the development of an effective control measure in the
future.

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Author contributions
Methodology: S.D., C.S. Software: C.S. Validation: T.L.T., N.B. Formal analysis: S.D., N.H.H. Investigation:
N.H.H. Resources: C.S., N.K.P. Data curation: N.K.P. Writing-original draft: R.S. Writing-review & editing: R.S.,
P.P. Visualization: T.L.T. Supervision: P.P. Project administration: N.B. All co-authors reviewed the final version
and approved the manuscript before submission.

Acknowledgements
Authors wish to express the deepest thanks to the SUT Research and Development Fund, the Suranaree University of
Technology for providing the grant support. We would like to express thanks to Dr. Sopone Wongkaew for their
kindness, instructions and assistance. Also, this research is partially supported by funds from the National Research
Council of Thailand and CS Tapioca Research and Innovation Co., Ltd. under the support of iTAP program from the
National Science and Technology Development Agency, Thailand. This work was supported by (i) Suranaree
University of Technology (SUT), (ii) Thailand Science Research and Innovation (TSRI), and (iii) National Science,
Research and Innovation Fund (NSRF) (project code 42850).

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