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MycoAsia – Journal of modern mycology ISSN: 2582-7278
www.mycoasia.org | 2022/06 1
Fruit brown rot caused by Neoscytalidium dimidiatum on Selenicereus monacanthus in
the Philippines
Mark Angelo Balendres1, *, John Darby Taguiam1, Edzel Evallo1, Jaypee Estigoy2, Cris Cortaga1
1Institute of Plant Breeding, College of Agriculture and Food Science, University of the Philippines Los Baños, Laguna,
Philippines 4031. 2Agricultural Science and Technology School, Central Luzon State University, Science City of Muñoz,
Nueva Ecija, Philippines 3120.
*Correspondence: mobalendres@up.edu.ph
Abstract
Multiple fungal pathogens infect economically important fruits, thereby affecting their quality and
marketability. Previous research showed that some fungal pathogens that can infect the stems might
infect the fruit but show a different symptom. To determine the causal pathogen of a fruit disease of
Selenicereus monacanthus (Dragon fruit), we used a combination of fungal pathology characterization
and molecular biology techniques. This paper presents the pathogenicity of Neoscytalidium
dimidiatum MBDF36C to S. monacanthus resulting in brown rot and canker on fruits and stem,
respectively. The paper also demonstrates the in vitro inhibition of N. dimidiatum MBDF36C by
chemicals, including a bio-fungicide containing Bacillus subtilis. At seven days post-inoculation, we
observed severe browning on N. dimidiatum MBDF36C-inoculated fruits but not on stems. Stems
exhibited canker-like symptoms. The same fungus was re-isolated from both inoculated diseased fruits
and stems, thereby confirming Koch's postulates. The pathogen was identified as N. dimidiatum based
on its morphology, cultural characteristics, and sequences of the partial ß-tubulin gene. In vitro growth
of N. dimidiatum MBDF36C was also completely inhibited by a bio-fungicide containing B. subtilis,
isoprothiolane, and mancozeb. This study is the first report of N. dimidiatum causing brown fruit rot
of dragon fruit in the Philippines. This information could impact the current postharvest fruit handling
operations and future studies on dragon fruit disease management.
Keywords: Bacillus subtilis QST strain 713, brown rot, dragon fruit, fruit disease, stem canker
Balendres MA, Taguiam JD, Evallo E, Estigoy J, Cortaga C (2022) Fruit brown rot caused by Neoscytalidium dimidiatum
on Selenicereus monacanthus in the Philippines. MycoAsia 2022/06.
Received: 17.02.2022 | Accepted: 30.06.2022 | Published: 15.07.2022
Handling Editor: Dr. Belle Damodara Shenoy
Reviewers: Dr. Tamie C. Solpot, Dr. Thomas Edison E. dela Cruz
Introduction
Tropical crops are famous for their delicious, water-rich taste and as a cash crop. Therefore, the market
is expanding. Many countries such as the Philippines, where these tropical crops are grown, benefit
from the global export market. While banana, pineapple, and papaya are the three more commonly
exported fruits, minor but emerging tropical fruits are beginning to penetrate the global market. One
of which is the edible cactus known as dragon fruit or Pitahaya (Selenicereus species) (Casas and
Barbera 2002, Nobel 2002). While the dragon fruit market is relatively small compared to banana and
pineapple, this budding industry is steadily and increasingly becoming popular. Vietnam is leading the
world's dragon fruit export (Mercado-Silva 2018, Tel Zur 2015). Other Southeast Asian countries
follow Vietnam's track (Balendres and Bengoa 2019). In the Philippines, the dragon fruit industry over
the past decade has grown (Eusebio and Alaban 2018). From a small 12-hectare farm in the early
1990s, the area planted with dragon fruit has reached almost 600 hectares with a volume of production
of more than 1800 mt in 2020 (Philippine Statistics Authority 2021). Dragon fruits are consumed as
fresh or in various processed and preserved foods (Pascua et al. 2015). However, yield and quality
MycoAsia – Journal of modern mycology ISSN: 2582-7278
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limiting factors threaten the lucrative dragon fruit industry. In addition, diseases are a significant
concern (Balendres and Bengoa 2019).
There are 25 species of plant pathogens causing various diseases in dragon fruits, and more than 90 %
of these pathogens are fungi (Balendres and Bengoa 2019). Diseases caused by Neoscytalidium
dimidiatum (Penz.) Crous & Slippers proved to be problematic in the field once spots developed into
canker, leading to rotting and collapse of the stem (Chuang et al. 2012). Although stem diseases can
be destructive in the field, some plants can withstand the infection and produce fruit. Therefore, a more
pressing concern is understanding when the pathogens infect the fruits. Diseased fruits, e.g., those
showing rot and spots, could be devalued because of their appearance or, worst, rejected by buyers or
consumers. Diseased fruits may be rejected during export due to the biosecurity rules of the importing
countries. The common fungal pathogens that attack dragon fruits are Bipolaris cactivora (Taba et al.
2007, Tarnowski et al. 2010) and N. dimidiatum (Lan et al. 2012, Ezra et al. 2013, Yi et al. 2015), but
no fruit disease has yet been scientifically reported in the Philippines. Fruit diseases are better
addressed when the cause of the disease is identified.
Chemicals have been used to control major dragon fruit diseases, including those caused by N.
dimidiatum (Balendres and Bengoa 2019), but given the growing concern over the use of synthetic
pesticides and some preference of consumers to organically grown fruits, biopesticides, alongside field
hygiene practices, are likely the preferred disease control management. However, some biopesticides
can have a phytotoxic effect on fruits (Aifaa et al. 2013). Biopesticides based on microbial antagonists
may fill this gap. Bacillus subtilis strains have been used against Bipolaris cactivora (Bae et al. 2013)
and on anthracnose pathogens (Meetum et al. 2017) of dragon fruits. However, reports on large-scale
commercial applications are scarce. Testing commercially available biofungicides could expedite the
selection of relatively safer chemicals for disease management of dragon fruit.
A diseased fruit of the red-skin, red-fleshed dragon fruit (S. monacanthus) from San Juan, Science City
of Muñoz, Nueva Ecija, Philippines, was submitted to the laboratory for diagnosis and subsequently
to identify the causative agent. Anecdotal claims of fruit diseases in dragon fruits in the country have
been made, but are not documented. The disease symptoms have also been linked to fungal pathogens,
as reported in the literature. Nevertheless, no empirical evidence to support such claims has been
presented. Moreover, insect pests could damage the fruit, and these insect-damaged tissues could be
erroneously identified as a fruit disease. This study aimed to isolate and characterize the causative
agent of the diseased fruit using combined morpho-cultural and molecular characterization methods.
Materials and Methods
Source of diseased fruit and fungal isolation
The diseased fruit of S. monacanthus, showing complex symptoms ranging from white to brown spots,
canker-like lesions, browning, and rot, was obtained from San Juan, Science City of Muñoz (15.7295°
N, 120.8729° E), Philippines. Fungi were isolated following the method of Balendres et al. (2020).
Briefly, infected-fruit tissues (2×2 mm in size) were sterilized in 10 % Sodium hypochlorite solution
(v/v, Zonrox, GreenCross Philippines), then washed three times with sterile distilled water at 1 min
and 30 sec each. Fruit tissue-cuttings were then blot-dried in sterile tissue paper. Once dried, fruit
tissue-cuttings were plated in potato dextrose agar (PDA) medium (HiMedia Laboratories Pvt Ltd,
India) and incubated at 28 °C for 7 days. Five fungal isolates (designated as Iso1 to Iso5) were obtained
that differed in cultural growth in the PDA medium. However, the pathogenicity assay (see the section
below) indicated that only Iso5 was consistently causing disease on the test fruits. Hence, only Iso5
was further assessed and studied. The fungus (designated as isolate MBDF36C) was deposited at the
Fungal Repository of the Plant Pathology Laboratory, Institute of Plant Breeding (IPB), College of
Agriculture, and Foods Science, University of the Los Baños, Laguna, Philippines.
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Morphological and Cultural Characterizations
A pure culture of isolate MBDF36C was obtained and subsequently grown in PDA medium. The
fungal morphology and cultural characteristics were assessed following the procedure used by
Balendres et al. (2020).
Molecular Identification
Fungal genomic DNA was extracted using the procedure of Doyle and Doyle (1987) and Cullings
(1992). The DNA sequence of the partial ß-tubulin gene was amplified following the Polymerase
Chain Reaction (PCR) procedure used by Dela Cueva et al. (2018) using primer pair, T1 (5′ AAC ATG
CGT GAG ATT GTA AGT) and T22 (5′ TCT GGA TGT TGT TGG GAA TCC) (O'Donnell and
Cigelnik 1997). The amplification started with a 3 min initial denaturation at 95 °C, followed by 30
cycles of 94 °C for 1 min, 52 °C for 30 s, 72 °C for 1 min, and a final extension step of 72 °C for 10
min (Dela Cueva et al. 2018). The PCR product was sent to Apical Scientific Sdn. Bhd. (Malaysia) for
sequencing and the consensus DNA sequence was then derived from the forward and reverse DNA
sequences using the sequence editing software Geneious R9 (Biomatters, New Zealand). First, to
identify the pathogen based on % sequence identity, an analysis was performed using the BLASTN
program (Zhang et al. 2000) to determine the closest fungal species. A phylogenetic tree was then
constructed using the Maximum Likelihood (ML) method based on the T92 (Tamura-3) parameter
(Tamura and Nei 1993) with uniform rates of nucleotide substitution supported with 1000 bootstrap
replicates (Felsenstein 1985). The analysis was conducted in MEGA version 7 (Kumar et al. 2016).
Neofusicoccum parvarum CBS 112930 (GenBank No. KX464983) was used as an outgroup.
Pathogenicity Assay
Ripe fruits and healthy young stem cuttings of S. monacanthus used in the pathogenicity assays were
sourced from the dragon fruits plants maintained at the IPB. These fruits and stem cuttings were not
sprayed with any chemicals. In the detached fruit assay, fruits were washed with tap water, surface-
sterilized in commercially available 10 % Sodium hypochlorite solution (v/v, Zonrox, Green Cross,
Philippines), and washed three times with distilled water before air drying. Fruits were then placed in
a plastic container overlaid with moist tissue paper. Three replicate fruits were used. Each fruit had six
wound-inoculated sites (pricked using pipette tip), corresponding to 5 fungal treatments and a distilled
water control treatment. 10 µl of spore suspension (>106 spores/ml) from seven-day old fungal cultures
was injected using a pipette into the inoculation sites. The container was sealed with the plastic cover,
and disease development was assessed seven days post-inoculation (dpi). In in vivo test, the
pathogenicity of N. dimidiatum MBDF36C was assessed on three-week-old rooted-stem cuttings of S.
monacanthus in the screenhouse. The same volume of spore suspension (prepared as above) was
inoculated on pre-determined wounded sites. The inoculated sites were then covered with transparent
tape to ward off insects or ants from entering the wound and avoid the spore suspension's rapid
evaporation. Wounded sites inoculated with distilled water served as the negative control. Disease
development was assessed at seven dpi. Re-isolation of the fungus from the diseased fruits and stems
was performed to establish Koch's postulates using the same method as described earlier.
In vitro Chemical Assay
The effect of a biofungicide containing either B. subtilis QST 713, isoprothiolane, mancozeb, citronella
oil or propamocarb was assessed in vitro using the poisoned food method (Grover and Moore 1962).
A five-mm mycelial plug of N. dimidiatum MBDF36C was placed at the center of the PDA medium
(in Petri plates) containing recommended rates of the test chemicals (Table 1). Pyraclostrobin (Xu et
al. 2018) was used as the chemical control. The PDA medium with sterile distilled water only (no
chemical) was used as the negative control. The trial was replicated three times. Mycelial growth was
measured at three dpi when the fungal growth in the negative control reached the edge of the plate.
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The mean percent inhibition was computed, and data were analyzed by the variance (ANOVA) test.
Multiple comparisons of means were carried out using Tukey's HSD test.
Table 1. Mean percent growth inhibition of Neoscytalidium dimidiatum MBDF36C in potato dextrose agar (PDA) medium
amended with sterile distilled water (negative control) and various chemicals at three days post-incubation.
Chemical Treatment
Rate Used1
Mean % Growth Inhibition2
Pyraclostrobin (chemical control)
1 mL
100 (±0.00)a
Bacillus subtilis strain QST 713
2 mL
100 (±0.00)a
Isoprothiolane
2.25 mL
100 (±0.00)a
Mancozeb
2 g
100 (±0.00)a
Citronella oil3
1.25 µl
72.22 (0.79b
Propamocarb
1.6 mL
0 (±0.00)c
Distilled water (negative control)4
-
0 (±0.00)c
1Based on the recommended rate as per product's label and adjusted to rate per 400 mL dH20 (capacity of Schott bottle).
2As compared to the fungal growth in PDA medium sterile dH20, only in vitro food poison technique assay (see Materials
and Methods). 3Based on the rate used by Dela Cueva and Balendres (2018).4No chemicals added.
Values are mean±standard error of the mean. Means followed by the same letter within the column are not significantly
different at 0.05 level by Tukey's HSD test.
Results
Characteristics and Identity of MBDF36C
Isolate MBDF36C was fast-growing, reaching the edge of the PDA medium just after three days of
incubation. The arthroconidia were hyaline to dark brown (Figure 1a), thick-walled, one-celled, rod-
shaped, and occurred singly or in arthric chains. Initially, the fungus was white with dense white aerial
mycelium (Figure 1b) that gradually turned grey to dark with age, usually at seven days from culture
(Figure 1c). These morpho-cultural characteristics are those of Neoscytalidium dimidiatum. The
identity of fungal isolate MBDF36C was further confirmed by the amplification and sequencing of the
fungal TUB2 gene. First, the BLASTN search showed a 99.30 % sequence identity with several
isolates of N. dimidiatum that cause canker or die-back (KF778965, KC357307). The constructed
phylogenetic tree based on the ML method supported the identity of MBDF36C as N. dimidiatum
(Figure 2).
Pathogenicity of N. dimidiatum MBDF36C
Only N. dimidiatum MBDF36C caused consistent brown rotting symptoms in all three fruit replicates
(Figure 1d). Browning started at two dpi from the inoculated sites, which grew larger as days
progressed. Despite N. dimidiatium being a pathogen known to cause canker or die-back symptoms,
no canker-like symptom was observed in the inoculated and wounded sites. However, typical canker
symptoms developed at seven dpi on stems in the screen house. No disease or symptoms were
manifested in the distilled water-inoculated sites. The same fungus was re-isolated from the diseased
fruits and stems from the laboratory and screenhouse assays, thus confirming Koch's postulates.
Inhibition of N. dimidiatum MBDF36C Growth by Chemicals
Growth of N. dimidiatum MBDF36C in the PDA medium was significantly (P<0.005) and completely
inhibited by the biofungicide containing either B. subtilis, isoprothiolane, and mancozeb (Table 1),
which were assayed separately. Substantial and significant growth inhibition (72.22 %) was observed
in the PDA medium amended with citronella oil. Propamocarb was not effective against N. dimidiatum
MBDF36C (growth rate was similar to the control treatment at 90 mm in diameter). The effect of the
bio-fungicide containing B. subtilis, isoprothiolane, and mancozeb was comparable to that of
pyraclostrobin (chemical check).
MycoAsia – Journal of modern mycology ISSN: 2582-7278
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Figure 1. Morphology (a) and cultural characteristics of 3- (b) and 7-day old (c) Neoscytalidium dimidiatum MBDF36C
in potato dextrose agar (PDA) medium. Inoculation of the pathogen resulted in browning on fruits (d) and canker on the
stem (e) within the inoculation point (arrowed). No browning was observed on inoculation points with other fungi (Iso1 to
Iso4) and in control (ctl, distilled water) treatments.
Discussion
Bipolaris cactivora (Petr.) Alcorn (Taba et al. 2007, Tarnowski et al. 2010) and Neoscytalidium
dimidiatum (Lan et al. 2012, Ezra et al. 2013, Yi et al. 2015) have been reported to cause brown spots
and rot on fruits of Selenicereus monacanthus and S. undatus. In this study, fruit brown rot in S.
monacanthus was caused by N. dimidiatum MBDF36C. Black rot and brown rot have been previously
reported in Israel (Ezra et al. 2013) and China (Yi et al. 2015). Both diseases were found in S. undatus
(red skin, white flesh). Here, fruit browning is reported in the Philippines for the first time on the red
skin, white-fleshed, S. monacanthus. The causative agent, N. dimidiatum MBDF36C, also causes
canker on dragon fruit stems.
These morphological characteristics of N. dimidiatum MBDF36C were consistent with the description
of N. dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). This pathogen is also known as N.
hyalinum (Phillips et al. 2013). However, when Huang et al. (2016) described a related species, N.
novaehollandiae, the name of the former was changed back to the old name, which is N. dimidiatum.
Both, nonetheless, are phylogenetically related (Phillips et al. 2013). This pathogen, N. dimidiatum, is
notoriously known to cause stem canker on dragon fruits (Chuang et al. 2012, Hawa Mohd 2013, Xu
et al. 2018), resulting from the collapsing of the plant (Chuang et al. 2012). In this study, the N.
dimidiatum MBDF36C isolates can also infect the stem and cause canker-like symptoms in S.
monacanthus. The result suggests that the inoculum during fruit infection likely came from infected
stems. Indeed, N. dimidiatum can also cause a canker on fruits (Sanahuja et al. 2016, Taguiam et al.
2020).
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Figure 2. Phylogenetic analysis by Maximum Likelihood (ML) method using the TUB2 sequence of Neoscytalidium
dimidiatum MBDF36C (arrowed) Philippine isolate (red arrowhead) and other N. dimidiatum (syn. N. hyaline) isolates.
The evolutionary history was inferred using the ML method based on the T92 (Tamura-3) parameter (Tamura and Nei
1993) with uniform rates of nucleotide substitution supported with 1000 bootstrap replicates (Felsenstein 1985). The
analysis was conducted in MEGA version 7 (Kumar et al. 2016). The tree was rooted to Neofusicoccum parvarum CBS
112930 (GenBank No. KX464983).
Chemicals are used to mitigate severe infection of dragon fruit diseases in the field. Nevertheless,
information on what chemicals and their effectivity are not widely reported. Azoxystrobin and
difenoconazole have been shown to control anthracnose and stem canker (Noegrohati et al. 2019).
Hexaconazole, tebuconazole, flusilazole, and pyraclostrobin inhibit N. dimidiatum growth (Xu et al.
2018). These chemicals are known to suppress the spore germination and fungal growth either by
affecting the fungal cell walls or inhibiting mitochondrial respiration. In this study, isoprothiolane and
mancozeb completely inhibited the growth of N. dimidiatum MBDF36C. These results were
comparable to pyraclostrobin and corroborated with the previous findings of Xu et al. (2018). While
the result promises chemicals that can be used for control, it is not without a disadvantage from an
environmental and health perspective. Therefore, attempts have been made to select a relatively eco-
friendly approach, e.g., biopesticides in the form of essential oils.
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Cymbopogon essential oil has been previously used to control dragon fruit anthracnose (Aifaa et al.
2013). However, essential oil higher than 2 % was phytotoxic and was not recommended for treatment.
Why a relatively higher concentration is phytotoxic remains unknown but it might have something to
do with the complex interactions of multiple chemical components in the essential oil that need to be
characterized (Brokl et al. 2013). In this study, N. dimidiatum MBDF36C growth was strongly
inhibited by citronella essential oil at a 1.25 uL/mL concentration. In another study, Dela Cueva and
Balendres (2018) found that citronella essential oil higher than 1.25 uL/mL concentration, although
helpful in mitigating anthracnose symptoms, can negatively affect the quality of pepper fruits. Thus,
future studies involving citronella essential oil in dragon fruit disease management would need to
include the effect of the oil on the quality of the fruits and stems. Biological control agents, e.g.,
Bacillus subtilis, have also been shown to inhibit fungal growth. They can also reduce the virulence of
anthracnose pathogens (Meetum et al. 2017). In this study, a bio-fungicide containing B. subtilis QST
713 was found to inhibit the in vitro growth of N. dimidiatum MBDF36C completely. The inhibitory-
growth effect was comparable to the chemical check, pyraclostrobin. However, the in vitro assay may
not reflect the actual efficacy of the biofungicide in the field. Hence, future screenhouse and field
studies would need to incorporate the comparison of rate, timing, and frequency of biofungicide
application. Information could lead to a better understanding of how to maximize the use of the
biofungicide to manage diseases (stem canker and fruit brown rot) caused by N. dimidiatum.
Propamocarb did not inhibit the growth of the pathogen and the mechanism is yet to be determined.
In conclusion, Neoscytalidium dimidiatum MBDF36C causes fruit rotting and stem canker in dragon
fruit. The fungal pathogen was identified using morphological, cultural, pathogenicity, and molecular
characterization. This study also demonstrates the potential of the biofungicide containing B. subtilis
in managing diseases caused by N. dimidiatium. However, further screenhouse and field trials are
warranted to further underpin the chemical's efficacy.
Acknowledgments
We thank Fe Dela Cueva and Vangelene Linga for technical assistance in the laboratory. We also thank
Ryan Tiongco, Joseph Lagman, and Anthony Vicencio for assistance in the screen house. Jennelyn
Bengoa and Rodel Maghirang kindly provided fruits and stems of dragon fruits. This study was
supported by a grant from the Department of Agriculture-Bureau of Agricultural Research (N825921)
awarded to M.A. Balendres. In addition, the Institute of Plant Breeding, College of Agriculture and
Food Science, University of the Philippines Los Baños, and the DOST-SEI have provided in-kind
support.
Statement on conflict of interest
The authors declare that they have no conflict of interest.
Authors contribution
MAB wrote the manuscript, analyzed the data, conceived and designed the research study. JDT and
EE conducted the experiments, analyzed the data, and co-wrote the manuscript. JE and CC collected
the samples and co-wrote the manuscript.
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