INTRODUCTION
Dragon fruit, known as pitaya is an imported crop from Vietnam and Taiwan. The fruit is widely cultivated in different areas of Thailand such as Buri Lam, Kon Kaen, Nakhon Ratchasima and Loei provinceatthenorth eastern part, Chanthaburi, Rayong, and Chon Buri province at the eastern part, Nakhon Pathom and Samut Sakhon at the middle part, while the western part is located at the Ratchaburi province. Many farmers who grow fruit trees in the northern part such as Chiang Mai, Chiang Rai and Phayao provinces find this fruit to be interesting. They find pitaya is a simple plant to grow and to manage in the field, and also has high yield. The fruit is a rich source of vitamins and nutrients for good health. It has been found to help the digestive process due to its fiber. It also prevents colon cancer and diabetes, neutralizes toxic substances such as heavy metals, and helps to reduce cholesterol levels and high blood pressure (He et al., 2012; Zainoldin and Baba, 2009). However, the main problem of pitaya production is plant diseases which could reduce yield both in terms of quantity and quality. The diseases have been found to attack the crop in both the pre-harvest and post-harvest stages. Several diseases have been reported on pitaya in the tropical and subtropical countries such as anthracnose (Colletotrichum spp.) (Palmater et al., 2007; Guo et al., 2014; Masyahit et al., 2013 and Lin et al., 2017), wilt disease (Fusarium oxysporium), stem rot (F. semitectum, F. oxysporium, F. moniliforme, F. fujiduroi (Hawa et al., 2012)), stem blight (Diplodia sp., Ascochyto sp. and Phoma sp.), soft rot (Erwinia sp., Enterobacter cloacae), black spot (Alternaria sp.), speck blight (Nectriella sp.) (Wang et al., 2007), brown spot (Botryodiplodia sp.), basal rot (Pytium sp.) (Lin et al., 2006), stem rot (Fusarium solani) (Rita et al., 2013) and brown spot diseases (Botryosphaeria dothidea) (Valencia-Botin et al, 2013). Fruit rot disease caused by Bipolaris cactivora, was found to be the most serious disease at the postharvest stage (He et al., 2012; Oeurn et al., 2016). Especially, Thailand weather is suitable for the epidemic diseases in the field. Suhi-Aromna (2015) reported that there are three major diseases of pitaya in Thailand such as anthracnose caused by C. gloeosporioides and C. truncatun are found to infect stems and fruits, fruit rot caused by Bipolaris cactivora (Oeurn et al., 2015) caused damage to stems and fruits, and brown spot or stem canker caused by Neoscytalidium dimidiatum (Thongkam and Soytong, 2016) was severely damaged pitaya crop production. Chemical pesticides are commonly used to control the diseases in the planting plot in Thailand. Samples of these chemical pesticides are copper oxychloride, azoxystrobin, difenoconazole, propiconazole, procloraz and mancozeb (Sudhi-Aromna, 2015). Recently, consumers have realized that there are chemical residues in agricultural products, especially the fresh ones. Furthermore, chemical pesticides are affecting both the farmer and his environment. Integrated Pest Management (IPM) was used to control pests and diseases in the planting plots. IPM tries to reduce chemical pesticides by using bio-pesticides as a substitute to chemicals which are alternative disease control method and useful in the development of more sustainable strategies in disease management in crop production. The objective of this study was to introduce the control techniques on pitaya diseases in the field (stem rot caused by Botryosphaeria dothidea) and postharvest disease (fruit rot caused by Bipolaris cactivora ) by using bio-pesticide and substitute chemicals in the northern part of Thailand.
MATERIALS AND METHODS
Survey and collecting plant diseases
A survey of major diseases of pitaya fruit at major planting areas which are in the highlands of the northern part of Thailand was conducted in 2014-2015. The infected plants were collected from three provinces including Chiang Mai, ChiangRaiand Phayao provinces. The samples were photographed to record the symptoms and put them in labeled plastic bags. The sample bags were kept in an ice box during transportation to the laboratory.
Experiment of fruit rot disease
Collecting, isolation and pathogenicity test
Collecting the infected fruits from the fresh market such as Ton Payom, Varorot, and Muang Mai markets, inChiangMaicity was conducted from June 2014-April 2015. Observation and recording of the symptom was made. Pure isolation was done by singer spores and were isolated with a sterile needle under a stereo microscope at 200x magnification and cultured on potato dextrose agar (PDA), incubated at room temperature for three days. Mycelium after three days of incubation was transferred to a new PDA to obtain pure fungal culture. Then, transferring the pure isolation was done in the test tube to be the stock culture for using the next experiment.
Inoculation method and pathogenicity test
The healthy pitaya fruit was considered by using identical fruit weight. It was found to have no damage from pests and diseases. The fruit that was used in this experiment was disinfected thoroughly with water, followed by surface disinfectant with 70% ethanol and was air-dried before application. Comparison inoculation with three kinds of methods as spraying anddroppingwithspore suspension, and placing with culture disc on the fruit were made. For spraying and dropping, B. cactivora solution was prepared spore suspension and was adjusted to 1x106 spores/ml, using a haemacytomether. Wound inoculation was done in each experiment comparing with control treatment (no wound). The treatment was created by a 2 mm deep puncture for 5-point using a sterile needle. The treated fruits were incubated in the moist plastic box at room temperature. Three replications were maintained for each isolate. Lesion diameters were measured after two and four days after inoculation.
Pathogenicitytestwere done after having the suitable method to inoculate the pathogen on fruit. There is a record period time on the appearance of a disease and diameter of lesion to select the virulent isolate for next experiment.
Efficacy test
Effect of wood vinegar on mycelium inhibition of fruit rot pathogen
The wood vinegar was derived from two kinds of woods including para rubber and eucalyptus wood. Poison media prepared with mixed each concentrations of wood vinegar in PDA at 4 concentrations as 0.5, 1.0, 1.5, and 2.0% (v/v). Mycelia discs (0.5 mm diameters) from the peripheral region of a7 day B.cactovora culture which was grown on PDA, and transferred onto the poison plates (9 cm) and PDA plate (control treatment). The plates were then incubated at room temperature. Three replications were maintained for each isolate. Zone inhibition of fungus was calculated using this formula:
PI = (R0-R1) /100 x 100
PI = Percentage of inhibition (%)
Ro = Fungi mycelia growth toward margin plate of control treatment (mm)
R1 = Fungi mycelia growth toward margin plate of treatment (mm)
Effect of wood vinegar on spore germination of fruit rot pathogen
Slide culture technique was used to check characteristic and germination of B. cactivora. The pathogen solution was prepared in spore suspension which was adjusted to 1x104 spores/ml, using a haemacytomether. The 1 ml of spore suspension was spread and cultured on the PDA which was mixed with 4 concentrations of wood vinegar at 0.5, 1.0, 1.5 and 2.0% (v/v). It was then placed in a piece of agar which was cut at 1x1 cm2 from plate on the slide, and incubated at room temperature. Data record was done as follows: 0, 3, 6, 9, 12 and 24 hr, respectively, to check the spore germination.
Effect of wood vinegar on control fruit rot disease in pitaya fruit
The healthy pitaya fruit was sterilized by surface disinfectant with 70% ethanol and air-dried before application. The experiment was done by 10 treatments as showed in Table 1. The treated fruits were incubated in the moist plastic box at room temperature. The treatment of this experiment was replicated four times. Lesion diameters were measured at two and four days after testing.
Table 1. Conditions of an effect on wood vinegar on control of fruit rot disease in the laboratory
Treatments |
Conditions1/ |
C1 |
Soaked in sterile water |
C2 |
Soaked in sterile water + placed pathogen |
C3 |
Soaked in 1.0% of wood vinegar for 1 min |
C4 |
Soaked in 1.0% of wood vinegar for 3 min |
C5 |
Soaked in 1.5% of wood vinegar for 1 min |
C6 |
Soaked in 1.3% of wood vinegar for 3 min |
T1 |
Soaked in 1.0% of wood vinegar for 1 min + placed pathogen |
T2 |
Soaked in 1.0% of wood vinegar for 3 min + placed pathogen |
T3 |
Soaked in 1.5% of wood vinegar for 1 min + placed pathogen |
T4 |
Soaked in 1.5% of wood vinegar for 3 min + placed pathogen |
1/ 4 replications/treatment
Stem rot disease
Collecting and isolation
Collecting the infected stem from the major planting plots of pitaya at three provinces including Chiang Mai, ChiangRaiand Phayao provinces was conducted. Pure isolation was done by singer spores as described above.
Dual culture test
The antagonistic activity of SOL-G3 (Bacillusamyloliquefaciens subsp. plantarum) bio-product which was derived from Plant Protection Center (PPC) research project belongs to the TaiwanICDF project between 2014 and 2019, against the stem rot pathogen that were tested by dual culture technique. Antagonistic bacterial was streaked at one side of PDA plate (2 cm away from the plate margin). Culture disc (5 mm) of fungal pathogen was cut with a sterilized cork borer from the margin of three days old culture and placed on the PDA opposite side of plate perpendicular to the bacterial streak respectively and incubated at room temperature for five to seven days. The PDA plates were placed with fungal discs alone and served as control. Three replications were maintained for each isolate. Observations on the width of inhibition zone and mycelia growth of test pathogen were recorded and percentage inhibition of pathogen growth was calculated with this following formula as described above.
RESULTS AND DISCUSSIONS
Survey and collecting plant diseases
A survey was carried out in the pitaya plantation in the highland of northern Thailand. Common diseases were found to have four major diseases in the planting areas e.g. stem rot caused by bacteria and caused by fungi; anthracnose, and brown spot or stem spot diseases. Fruit rot disease was found to be the most serious diseaseatthepostharvest stage. In this research, the focus was on fruit rot and stem rot diseases and the use of chemical substitutes and bio-pesticide to control both diseases.
Experiment of fruit rot disease
Collection and isolation
A fruit rot disease was observed in white-fleshed dragon fruit which was collected from the fresh market in Chiang Mai city. Common symptoms on the surface of the fruits were brown spots with water soaked and black powdery spots, circular lesionontheskin fruit (Fig. 1A). Dark grey to dark green cottony mycelia were observed on PDA for seven days (1C). The morphology under light microscope at 400x magnification of B. cactivora on fruit, conidiophore was light brown to dark brown, straight or flexuous. Conidia were straight, ellipsoidal, fusiform or ovate, 3-6 septa, pale light brown to brown, and conidia size were 4.88 – 7.32 (-9.76) × (24.4-) 34.16 – 48.8 (-61) µm in length (Fig. 1E and F4). This result was similar to report of He et al. (2012) and Oeurn et al. (2015) were reported that conidia of pathogen were fusiform and obclavate, light brown to brown conidia, 3-5 septa. Ten fungal isolates were obtained including five isolates from Varorot market, three isolates from Ton Payom market, and two isolates from Muang Mai market. Colony on PDA appeared dark green to black color, mycelia were fluffy on PDA (Fig. 1C).

Fig. 1. Morphology of B.cactovora causal agent of pitaya fruit rot: (A) disease symptom on fruit, (B) colonies of conidia on fruit, (C) characteristic of pathogen colony on PDA at 7 days, (D) mycelia and conidia, and (E and F) of pathogen under light microscope at 400x magnification.
Inoculation method and pathogenicity test
Using spore suspension of B.cactivora inoculated with spraying method on the fruit resulted in a healthy fruit (for the control group) and having a wound (from the treatment). The result showed the disease occurred on the fruit which had the lesion with light brown spot at two days after inoculation. After that, the disease appeared as black color spore covering the lesion at four days after inoculation. Comparing with the control treatment which no lesion, no disease has been found on the fruit (Fig. 2).

Fig. 2. The fruit rot disease revealed on the fruit which was sprayed with spore suspension of B.cactivora at four days after inoculation: (C1) control treatment: no wound and sprayed with sterile water, (C2) had wound and sprayed with spore suspension, (T1) no wound and sprayed with spore suspension and (T2) made wound and sprayed with spore suspension.
Using spore suspension of B.cactivora inoculated with dropping method on the fruit which resulted in the wound and no wound on fruit. The result showed the small brown spot was revealed on fruit which resulted in a wound two days after inoculation. At four days after inoculation brown spots was found on the fruit with water soak and black color of spore covering the lesion after four days of inoculation (Fig. 3).

Fig. 3. The fruit rot disease revealed on the fruit which wasdroppedwithspore suspension of B. cactivora at four days after inoculation: (C1) control treatment: no lesion and sprayed with sterile water, and (T2) made wound and sprayed with spore suspension.
Using culture disc of B.cactivora was inoculated by placing on the fruit which resulted in wound and no wound on fruit. The result showed the tissue changed from light brown to brown small lesion with water soak was revealed on fruit which had the lesion at two days after inoculation. At four days after inoculation typical lesion was found on the fruit including black color of spore covering the lesion after four days of inoculation (Fig. 4).

Fig. 4. The fruit rot disease revealed on the fruit which placed culture disc of B.cactivora after four days of inoculation: (C1) control treatment: placed PDA disc on fruit, and (T2) placed culture disc on fruit.
Inoculation with placed culturediscofpathogen on the fruit was used in the next experiment because the result showed typical symptoms. Pathogenicity test was used in culture disc of various isolates of B. cactivora which was placed on the fruit. The result revealed all isolates caused the disease of fruit similar to a previous experiment and report of He et al. (2012).
Efficacy test
Effect of wood vinegar on mycelia inhibition of fruit rot pathogen
Five isolates of B.cactivora including BcDFW_2, BcDFW_3, BcDFW_4 and BcDFW_5 were tested with wood vinegar at concentration of 0.5, 1.0, 1.5 and 2.0% (v/v) were tested for the mycelia inhibition. The result showed that para rubber wood vinegar gave significantly higher in mycelia inhibition of pathogen compared to eucalyptus wood vinegar. It revealed that para rubber wood vinegar had 100% of mycelia inhibition at concentration of 1.5 and 2.0% (v/v) in all isolates of the pathogen, while eucalyptus wood vinegar had a range of 50.37 - 60.37% and 85.19 – 100% at concentration of 1.5 and 2.0% (v/v) in all isolates of the pathogen, respectively which is significantly at 95% level (LSD p=0.05) as showed in Table 2.
Table 2. Effect of para rubber and eucalyptus wood vinegar on mycelia inhibition of B.cactivora caused of pitaya fruit rot on PDA at 7 days after incubation.
Kinds of wood vinegar |
Isolates of fungi |
Percentage of mycelia inhibition1/ |
1.0 % |
1.5 % |
2.0 % |
1.0 % |
Para rubber |
BcDFW_2 |
42.78fghi 2/ |
67.41cd |
100a |
100a |
|
BcDFW_3 |
39.26hij |
68.15cd |
100a |
100a |
|
BcDFW_4 |
36.67hijk |
97.04a |
100a |
100a |
|
BcDFW_5 |
36.67hijk |
68.89c |
100a |
100a |
eucalyptus |
BcDFW_2 |
34.44ijk |
41.11ghi |
50.37efg |
85.19b |
|
BcDFW_3 |
30.37jkl |
44.44fghi |
57.78de |
100a |
|
BcDFW_4 |
27.78kl |
42.96fghi |
60.37cde |
91.1ab |
|
BcDFW_5 |
22.96l |
45.19fgh |
52.96ef |
91.48ab |
LSD0.05 |
10.54 |
%CV |
9.68 |
1/ 3 replications/treatment
2/ Means followed by the same letter in each column are not statistically different by LSD (p=0.05)
Fig. 5. Effect of wood vinegar at concentration of 0.5, 1.0, 1.5 and 2.0% (v/v) on mycelia inhibition of B. cactivora was test on PDA at seven days after incubation. (A) treatment of para rubber wood vinegar and (B) treatment of eucalyptus wood vinegar.
Effect of wood vinegar on spore germination of fruit rot pathogen
Effect of two kinds of wood vinegar on spore germination of B. cactivora was tested by slide culture technique with four concentrations of wood vinegar at 0.5, 1.0, 1.5 and 2.0% (v/v) at 3, 6, 9, 12 and 24 hours, respectively. The result showed that it found germ tube of the pathogen at three hours after incubation at control treatment and treatment as PDA witch mixed with concentration of eucalyptus wood vinegar at 0.5% (v/v), while it found germ tube at 9 hours after incubation at treatment as PDA which mixed with concentrations of para rubber wood vinegar at 0.5% (v/v) as 89.41% of mycelia inhibition. Moreover, no germ tube was revealed at the PDA which mixed with concentrations of wood vinegar at 1.0, 1.5 and 2.0% (v/v) as showed in Table 3.
Table 3. Effect of par rubber and eucalyptus wood vinegar on inhibition of spore germination of B. cactivora was tested on PDA at seven days after incubation.
Kinds of wood vinegar |
Concentration of wood vinegar |
Percentage of inhibition1/ (hr) |
3 |
6 |
9 |
12 |
24 |
Para rubber |
0.5 |
100a2/ |
100a |
89.41a |
87.05a |
83.65ab |
1.0 |
100a |
100a |
100a |
100a |
100a |
1.5 |
100a |
100a |
100a |
100a |
100a |
2.0 |
100a |
100a |
100a |
100a |
100a |
Eucalyptus |
0.5 |
46.47b |
27.05c |
13.49c |
1.96c |
1.04d |
1.0 |
100a |
74.64b |
69.92b |
64.67b |
55.89c |
1.5 |
100a |
100a |
90.42a |
82.95ab |
73.37bc |
2.0 |
100a |
100a |
100a |
100a |
100a |
LSD0.05 |
|
18.92 |
19.83 |
18.68 |
19.29 |
19.28 |
%CV |
|
13.89 |
15.49 |
15.44 |
16.61 |
17.22 |
1/ 3 replications/treatment
2/ Means followed by the same letter in each column are not statistically different by LSD (p=0.05)
Effect of wood vinegar on control fruit rot disease in pitaya fruit
Four isolates of B.cactivora including BcDFW_2, BcDFW_3, BcDFW_4 and BcDFW_5 were used. The para rubber wood vinegar at concentration of 1.0 and 1.5% (v/v) soaked the fruit for one and three minutes, were tested to control the fruit rot disease. The result showed that 1% concentration of para rubber wood vinegar for three minutes had highest percentage of disease inhibition at 89.29 - 94.53. It was found that 1.5% concentration of para rubber wood vinegar for one minute had percentage of disease inhibition at 77.08 - 100 as showed in Table 4.
Table 4. Effect on para rubber and eucalyptus woodvinegaratconcentration of 1.0 and 1.5% (v/v) fruit for one and three minutes on the control fruit rot disease in pitaya fruit.
Concentration of wood vinegar |
Soaking time |
Percentage of inhibition1/ |
BcDFW_2 |
BcDFW_2 |
BcDFW_2 |
BcDFW_2 |
Average 3/ |
1.0 |
1 |
100a 2/ |
68.75a |
92.86a |
79.1a 7 |
85.20a |
|
3 |
94.53ab |
91.67a |
89.29a |
93.06a |
92.14a |
1.5 |
1 |
100a |
77.08a |
78.57a |
93.06a |
87.18a |
|
3 |
84.37b |
100a |
71.43a |
75.70a |
82.88a |
LSD0.05 |
|
15.02 |
35.45 |
46.14 |
50.84 |
16.95 |
%CV |
|
10.29 |
27.27 |
36.07 |
38.71 |
12.67 |
1/4 replications/treatment
2/ Means followed by the same letter in each column are not statistically different by LSD (p=0.05)
3/Average of 4 isolates of the B. cactivora
Stem spot disease
Collecting and Isolation
A stem spot disease observed on the stem which was collected from pitaya planting plots in Chiang Mai, Chiang Rai and Phayao provinces in the northern part of Thailand. The early symptom was found with small and chlorotic spots on the stem that then developed into dark spots which included pycnidia structure inside the spot (Fig. 6A and 6B). Themorphologyunderlight microscope at 400x magnification of Botryosphaeria dothidea on the lesion. Conidia produced in pycnidia structure appeared on the dark spot (Fig. 6C). Conidia were straight and ovate, hyaline, one cell and conidia size were 5.48-9.53 × 1.57-3.15 μm in length (Fig. 6D). Colony on PDA were appeared dark gray to black color, mycelia were fluffy on PDA (Fig. 6E and 6F)
Fig. 6. Morphology of Botryosphaeria dothidea causal agent of pitaya stem spot disease: (A) disease symptom on stem, (B) lesion of stem spot disease under stereomicroscope at 400x magnification, (C) pycnidia of B. dothidea and (D) conidia under compound microscope at 400x magnification, (E and F) colonies on PDA at 6 days after incubation. \
Dual culture test
The result of the dual culture on PDA at six days after incubation showed that antagonistic bacterium, SOL-G3 (Bacillus amyloliquefaciens subsp. plantarum) inhibited growth of two isolates of tested fungi at average of inhibition percentage as 68.33 and 67.91, respectively (Table 5 and Fig. 7).
Table 5. Antagonistic activity of SOL-G3 (Bacillus amyloliquefaciens subsp. plantarum) against Botryosphaeria dothidea causal agent of pitaya stem spot disease in laboratory.
Isolate of fungi |
Replications |
Radial growth |
Percentage of inhibition |
Average of inhibition percentage |
1 |
1 |
1.9 |
68.33 |
68.33 |
|
2 |
1.8 |
70 |
|
|
3 |
2 |
66.66 |
|
|
4 |
1.9 |
68.33 |
|
2 |
1 |
1.9 |
68.33 |
67.91 |
|
2 |
1.8 |
70 |
|
|
3 |
2 |
66.66 |
|
|
4 |
2 |
66.66 |
|
Fig. 7. Antagonistic activity of SOL-G3 (Bacillus amyloliquefaciens subsp. plantarum) against Botryosphaeria dothidea causal agent of pitaya stem spot disease on PDA plate at 6 days after incubation.
In this research, the Integrated Pest Management (IPM) was used to control pests and diseases in the planting plots and also practiced the use bio-pesticides to reduce the use of chemical pesticides. Using IPM for control of diseases was applied on the field by the farmers who grow the crops. IPM is not a single control method but rather is a combination of management evaluations, decisions and controls. First, we promoted the principle of IPM method on how to control pests and diseases in the field and in each crop to the farmers in the GAP training program with at least a year of interval. Based on reduction of chemical residue in crop production or promotion of food safety by using bio-pesticide as alternative disease control method and the use of more sustainable strategies of disease management in crop production, a more sustainable agriculture can be achieved.
CONCLUSION
Based on the results, it can be concluded that there are four major diseases in the planting areas e.g. stem rot caused by bacteria and caused by fungi; anthracnose, and brown spot diseases. Fruit rot disease was found to be the most serious disease at the postharvest stage. Using substitute chemicals as pyroligneous acid or wood vinegar at concentrations soaking the fruits in wood vinegar solution at concentration of 1% (v/v) for three minutes was effective in the reduction of fruit rot disease of at 92.14% which is significantly at 95% level (LSD p=0.05). In addition, using the antagonistic bacterium, SOL-G3 (Bacillus amyloliquefaciens subsp. plantarum) inhibited growth rate of Botryosphaeria dothidea caused of stem rot in the laboratory, so further work will be tested in the field.
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