Pathogenicity of Epicoccum Sorghinum Towards Dragon Fruits
Pathogenicity of Epicoccum Sorghinum Towards Dragon Fruits
Pathogenicity of Epicoccum Sorghinum Towards Dragon Fruits
DOI: 10.1111/jph.12893
ORIGINAL ARTICLE
KEYWORDS
1 | I NTRO D U C TI O N due to the high-income potential as evident in the increase in area
devoted for dragon fruit (from 181.90 ha in 2012 to 449.50 ha in
Dragon fruit is a high-value crop that is grown worldwide. In the 2017), which consequently leads to more than 80% increase in the
Philippines, many growers have ventured into dragon fruit cultivation production (Eusebio & Alaban, 2018; PSA, 2018a, 2018b). However,
harvests in waves and decline in yield raise concerns of dragon 2.2 | Fungus identification
fruit shortage. Diseases are among the most important factors that
have contributed to the decline in the yield of dragon fruit world- Epicoccum sorghinum MBDF0024a was identified by using a com-
wide (Balendres & Bengoa, 2019). Diseases of dragon fruit in the bined morphological, cultural, and molecular identification tech-
Philippines are yet to be reported. niques (Cai et al., 2009). The fungus was transferred to a new PDA
Researches are uncovering the devastating effects of new and medium for 7 days to assess its growth in the media and its morphol-
emerging pathogens on plants or crops. Recent studies suggest that ogy under a light microscope. The form, colour, radial growth and
there are more dragon fruit diseases that are yet to be identified pigmentation of the mycelia were recorded at 7 days postincubation
and studied (Balendres & Bengoa, 2019). Emerging diseases may at the same condition as indicated above. Morphological features
play an important role in the rapid yield decline of dragon fruits and were examined under a microscope (Olympus CX22) and images
may affect the supply of stem cuttings which are sources of planting were analysed using the ImageJ software (Version 1.51s, Wayne
materials. Infected planting materials could assist in disease spread Rasband, National Institutes of Health). The morphology and cul-
from one farm to another. Understanding of current and new patho- tural features of the fungus showed characters of an Epicoccum sp.
gens that infect dragon fruit is important in addressing production However, since there are other species within this genus, and mor-
issues caused by these pathogens. Identification of the pathogens pho-cultural features are highly variable to the growing environment
associated with the disease will also assist in formulating effective of the fungus, a molecular identification technique was further used.
and appropriate disease control measures.
In 2019, a diseased dragon fruit stem was observed in Los Baños,
Philippines. No disease is yet to be reported in dragon fruits in the 2.3 | PCR amplification and phylogenetic analysis
Philippines. The symptom did not resemble those commonly re-
ported in other countries, for example anthracnose and stem canker. The fungal genomic DNA of the isolate MBDF0024a was extracted
Hence, this study was aimed at identifying the pathogen associ- using a cetyltrimethylammonium bromide (CTAB) extraction proce-
ated with a stem disease of Hylocereusmonacanthus (pink-skin, red- dure (Cullings, 1992; Doyle & Doyle, 1987). The DNA was then used
fleshed). Because of the disease was not previously reported, this for the succeeding polymerase chain reaction (PCR) assay to amplify
study also screened potential chemicals with antifungal properties the internal transcribed spacer (ITS), beta tubulin (TUB2) and the actin
to the associated pathogen. (ACT) gene regions that will be used for subsequent DNA sequenc-
ing. For ITS, the PCR was performed in a MyCycler™ Thermal Cycler
(Bio-Rad, USA) in a 15-μl reaction volume, containing 1x PCR Buffer
2 | M ATE R I A L S A N D M E TH O DS (Invitrogen), 2.0 mM MgCl2 (Invitrogen), 0.2 mM dNTPs (Invitrogen),
0.2 μM each of the forward (ITS4, 5′-TCCTCCGCTTATTGATATGC-3′)
2.1 | Stem collection and fungal isolation and reverse (ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) prim-
ers (White, Bruns, Lee, & Taylor, 1990), 1 U Taq DNA Polymerase
Stems of pink-skin, red-fleshed, dragon fruit, H. monacanthus, that (Invitrogen), 2 μl of the fungal genomic DNA and DEPC water (Sigma)
have irregular-shaped, necrotic, brown lesions were collected from to volume. The thermal cycling conditions were as follows: initial de-
a dragon fruit plant grown in the research station of the Institute of naturation at 94°C for 5 min, followed by 24 cycles of denaturation
Plant Breeding (IPB), University of the Philippines Los Baños (UPLB) at 94°C for 45 s, annealing at 55°C for 45 s and extension at 72°C
(14°09′10.59′′N, 121°15′47.25′′E, 20 m above sea level) in Los for 1 min and a final extension at 72°C for 7 min. For TUB2 and
Baños, Laguna. The IPB houses germplasm accessions of the three ACT, the PCR assay was performed using the method of Dela Cueva,
commonly cultivated dragon fruit species (H. undatus, H. monacan- Mendoza, and Balendres (2018). All PCR product was resolved by
thus and H. megalanthus). gel electrophoresis [1.5% agarose (Vivantis) 0.5X Tris-acetate-EDTA
The fungus was isolated by cutting a healthy and infected buffer containing 2 μl GelRed solution (Biotium)] in PowerPac™ and
2
(lesion) portion of the stem tissues into 3 mm blocks. The small Sub-Cell GT (Bio-Rad Laboratories)], and the amplified product was
blocks were then surfaced sterilized in 10% sodium hypochlorite visualized using the GelDoc™ XR+ with Image Lab software (Bio-
(NaOCl) solution (v/v. Zonrox, Philippines) for 1 min, rinsed in ster- Rad Laboratories). The PCR product was sent to Apical Scientific
ile distilled water for three times, air-dried on sterile tissue paper Sdn. Bhd. (Malaysia) for DNA sequencing. The consensus DNA se-
and, finally, transferred or plated onto potato dextrose agar (PDA) quence was then derived from the resultant forward and reverse
medium (Himedia Laboratories Ltd., India). After 7 days, at room DNA sequences using the sequence editing software Geneious R9
temperature with 14 hr light in 24 hr cycle, 5 mm of the fungal (Biomatters, New Zealand). The consensus sequence of the ITS gene
mycelial plug was purified and transferred to a new PDA medium. region was compared with the DNA sequences of several Epicoccum
The same fungus grew from the sample dragon fruit stem tissues. species and other closely related species (Table 1) used by Aveskamp,
The isolated fungus was designated as isolate MBDF0024a and Gruyter, Woudenberg, Verkley, and Crous (2010). A distance tree
was deposited at the Fungal Repository of the Plant Pathology was produced using BLAST pairwise alignment (neighbour-joining
Laboratory, IPB, UPLB. tree method) with 1,000 bootstrap replication and was performed
TAGUIAM et al. |
3
Peyronellaea americana B CBS 185.85 FJ426972 proximately 1–2 mm) and unwounded sites. Sites inoculated with
sterile distilled water (SDW) and PDA medium plugs (without fungus)
Phoma boeremae T CBS 109.942 FJ426982
served as negative checks. Stems were placed in containers overlaid
Stagonosporopsis lupini B CBS 101.494 GU237724
with wet tissue paper (to provide humidity), and covered with plastic
a
T: Ex-type strain; B: Reference strain according to Boerema, Gruyter,
bags and were kept at room temperature. Disease development was
Noordeloos, & Hamers (2004).
b recorded at 3 dpi. The detached stem assay was performed thrice,
CBS: Centraalbureau voor Schimmelcultures, Utrecht, The
Netherlands, PD: Plant Protection Service, Wageningen, the and each assay was replicated three times.
Netherlands. In the glasshouse assay, the fungus was inoculated on 3-week-
Source: Aveskamp et al. (2010). old rooted stem cuttings of Hylocereus undatus, H. monacanthus and
(a) (b)
H. megalanthus. Spore suspension and mycelial plugs were inoculated Petri plate. The in vitro chemical assay was performed twice, with each
as similarly described in the detached stem assay. Plants inoculated assay having three replicate plates. Data were analysed by analysis of
with SDW and PDA blocks (without fungus) served as the negative variance (ANOVA) tests, and means were compared using the LSD test.
checks. A transparent tape (approximately 2 × 2 cm) was placed in
the wounded area to cover the inoculated site. This avoided the
quick evaporation and the runoff of fungal spore suspension. The 3 | R E S U LT S
tape was removed at 4 dpi, and the inoculated site remained moist.
Thus, it was considered to be an effective method in retaining the 3.1 | Characteristics of Epicoccum sorghinum
spore suspension on the inoculated sites. The glasshouse assay was MBDF0024a
performed thrice. Three replicate plants per dragon fruit species,
with each plant having three inoculation sites, were used. Symptom The isolated fungus from diseased dragon fruit stem (Figure 1a)
development was assessed at 7 and 14 dpi. showed white to grayish mycelia with an average radial growth of
The fungus was re-isolated from infected stems in both detached 24 mm and 42 mm at 3 and 7 days postincubation, respectively. Pale
and glasshouse assays using the same procedure as described above. brown and globose conidia, measuring an average of 11.53 μm (30
conidia, ranging from 6.18 to 17.00 μm), were observed from the
7-day-old culture (Figure 1b). The fungus exhibited a reddish-brown
2.5 | Chemical assay pigmentation in the PDA medium at 7 days postincubation that
was clear at the underside of the Petri plate (Figure 1c,d). Culture
The response of the fungal isolate MBDF0024a to six chemicals (cit- and morphology characteristics resemble those of Epicoccum sp.
ronella essential oil, Bacillus subtilis, propamocarb, isoprothiolane, py- (Aveskamp et al., 2010). Using the primer pairs ITS4/ITSS, a 533
raclostrobin and mancozeb) was assessed by in vitro trials using the base pair product was amplified by PCR. The DNA sequence of the
poisoned food method (Grover & Moore, 1962). Chemicals at their ITS gene region of the fungus (isolate MBDF0024a) showed a 100%
recommended rate, according to the manufacturer, were amended similarity to E. sorghinum strains CBS 179.80 and CBS 627.68, based
separately to the PDA medium. The 1.25 µl/ml concentration of cit- on the BLAST search. Using the authentic sequences of Epicoccum
ronella oil was based on the test done by Dela Cueva and Balendres and other related species, the DNA sequence of the ITS gene of
(2018). Five-mm mycelial plugs of a fungal isolate of 7-day-old E. sorghi- isolate MBDF0024a was aligned with the E. sorghinum (Figure 2).
num MBDF0024a were placed at the centre of the chemical-amended The DNA sequences of TUB2 and ACT genes of MBDF0024a also
and non-amended PDA medium. Radial growth was assessed when showed 99.62% and 100% similarity, respectively, to E. sorghinum
the growth of the non-amended fungus has reached the edge of the strains PD76/1025 (FJ42782) and CBS 301.89 (FJ426957).
3.2 | Pathogenicity of Epicoccum sorghinum impact of dragon fruit diseases caused by E. sorghinum. While mul-
MBDF0024a to Hylocereus species tiple studies have reported several fungal diseases of dragon fruits
(see Balendres & Bengoa, 2019), this study is the first to report the
Epicoccum sorghinum MBDF0024a was pathogenic to the three pathogenicity of E. sorghinum to the three commonly cultivated
Hylocereus species in both the detached stems (Figure 3) and glass- dragon fruit species (H. monacanthus, H. undatus and H. megalan-
house trials (Figure 4). The disease developed as early as 3 dpi with thus), and the first to demonstrate that yellowing of the stem is also
sunken brown necrotic spots around the inoculated sites. The dis- caused by a fungus, not only by bacteria. Lastly, chemicals with in-
ease developed only on wounded sites regardless of the inoculation hibitory activity to E. sorghinum are demonstrated for the first time.
method (spore suspension or mycelial plugs) (Figure 5). However, The results of this study show that dragon fruit growers should
brown lesions developed in unwounded sites but only in the de- also pay attention to diseases, other than those frequently reported.
tached stem assay using the fungus’ mycelial plugs (Figure 3). The The most frequently reported fungal diseases are anthracnose caused
disease was severe in H. undatus (pink-skin, white-fleshed), then in by various Colletotrichum species (e.g. Meetum, Leksomboon, &
H. monacanthus (pink-skin, red-fleshed) and least in H. megalanthus Kanjanamaneesathian, 2015), stem canker caused by Neoscytalidium
(yellow skin, white-fleshed) (Figure 4). Yellowing of the lesion started
at 5 dpi, but in H. undatus, rapid yellowing of stem was observed
from 5 or 7 dpi in all glasshouse trials (Figure 4). All stems eventually
turned yellow 4 weeks postinoculation, expect in H. megalanthus.
No infection was observed in all negative checks. The fungus was
re-isolated from the diseased tissues, and the isolated fungus was
identical to the inoculated fungus.
The results of this study highlight three key informations that war-
rant further attention. First, the pathogen isolated from the diseased
stem of dragon fruit, Epicoccum sorghinum MBDF0024a, has not
been previously reported and none is known of its epidemiology and
management in dragon fruit. The findings further support the hy-
pothesis (Balendres & Bengoa, 2019) that there are more pathogens
associated with dragon fruit diseases that are yet unreported and
uninvestigated. Second, none of the commonly cultivated Hylocereus (d) (e) (f)
species were resistant to the stem disease caused by E. sorghinum, of
which after 7 days the disease can severely damage the stem, lead-
ing to collapse in succeeding weeks. In contrary to the commonly F I G U R E 3 Pathogenicity of Epicoccum sorghinum MBDF0024a
to wounded (W) and unwounded (UW) stems of Hylocereus
associated pathogens, which are bacteria, the fungus E. sorghinum
monacanthus (a and d), H. undatus (b and e) and H. megalanthus (c
MBDF0024 has been found to also induce yellowing of the stem.
and f) using mycelial plugs (upper photos) and spore suspension
Finally, as controls are not yet available, chemicals used in this study (lower photos) in detached stem assays
can be further used for field evaluation to mitigate the negative
|
6 TAGUIAM et al.
F I G U R E 4 Pathogenicity of Epicoccum sorghinum MBDF0024a to rooted stem cuttings of Hylocereus monacanthus, H. undatus and
H. megalanthus at 3 and 7 days postinoculation in glasshouse assays
Mean radial This study has demonstrated that Epicoccum sorghinum MBDF0024a
Treatment growth (mm)1 is pathogenic to the three commonly cultivated dragon fruits
PDA only (Control) 20 (0.45) a (Hylocereus species), resulting to severe stem disease. Further, op-
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