MINISTRY OF EDUCATION
MINISTRY OF AGRICULTURE
AND TRAINING
& RURAL DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENS

MAI VĂN TRỊ
STUDY ON THE CAUSAL AGENT AND CONTROL MEASURES
OF TRUNK CANKER ON JACKFRUIT
IN THE SOUTHEASTERN
MAI VĂN TRỊ

Major: PLANT PROTECTION
Code: 962122

SUMMARY OF THE DOCTORAL THESIS IN AGRICULTURE

Ho Chi Minh city - 2018


ii

.TS. NGUYỄN VĂN TUẤT
This thesis was completed at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

Supervisors: Prof. Dr. Nguyen Van Tuat
Dr. Nguyen Van Hoa
HÒA
Reviewer 1: ………………………………….
Reviewer 2: ………………………………....

1.3. The subject, location and duration of the study
The subjects were Phytophthora species causing trunk canker and
this disease on jackfruit in the Southeastern. Duration: 9/2012 – 3/2018.
1.4. Scope of study
This thesis focused on identifying Phytophthora species causing
jackfruit trunk canker and on studying several control measures to reduce
the disease intensity in the Southeastern.
1.5. Scientific and practical significance
This thesis provided scientific information on the trunk canker
disease and the causal agent (Phytophthora palmivora) on jackfruit. This


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thesis was a scientific background for the research and development of
strategies for integrated management of the soil-born Oomycetes pathogens.
These thesis results were scientific basis for building the procedure of
integrated management of trunk canker disease on jackfruit, were reference
source for research design and scope, for teaching and training materials.
These thesis results were going to be useful for in-field early
diagnosis and prompt treatment of the disease.
1.6. Significance of the study
- P. palmivora was identified as the causal agent of the jackfruit
trunk canker in the Southeastern region based on morphological
characteristics and the sequencing results of the rDNA-ITS region and the
COX II gene.
- Sequence of the rDNA-ITS region and COX II gene of the P.
palmivora causing jackfruit trunk disease were determined provided
scientific data for further studies on this important pathogen.
- Measures in reducing disease intensity were determined including:

in the Vietnam. Therefore, the study determining the causal agent and
developing measure strategies in reducing the impact of trunk canker is an
imperative need for the survival of the jackfruit industry.
More than 60 species of Phytophthora were recorded infesting
various crops worldwide [50]. Many species can be easily identified by
morphological techniques using a number of morphological and
physiological characteristics that were typically classified by Waterhouse
(1963) [151], Stamps et al (1990) [137] and Ho (1992) [77]. Besides
morphological techniques, molecular identification has been applied to
determine the species based on the internal transcriber spacers (ITS)
sequences of the ribosomal DNA. The combination between morphological
and molecular techniques has commonly used for identification
Phytophthora.
Each of the disease has its own characteristics, which makes it
difficult to generalize disease-control measures [59]. Therefore, it is


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important to understand the most common contributing factors that
underpin the control of Phytophthora diseases. Only an in-depth
understanding of these fundamental factors, coupled with a detailed
understanding of the agronomics of the crop will allow developing
effective, integrated disease control methods [47].
Effective disease control is rarely achieved through the application
of a single control measure [59] thus we need to use a number of integrated
approaches to limit the disease impacts. Integrated Pest Management (IPM)
or Integrated Disease Management (IDM) is the long-term reduction of
disease losses to economically acceptable levels through a holistic approach
that combines the use of resistant varieties, cultural control, biological

(BRVT) as well as in provinces of BRVT, Dong Nai, Binh Duong, and
Binh Phuoc of the Southeastern region.
2.3 Methodologies
2.3.1 Survey the status of jackfruit trunk canker
Survey was conducted form Jan., 2013 to Dec., 2014 in the orchards
not less than 1,500 m2 or 100 trees in the four provinces; according to
guidelines of National Technical Regulation on Surveillance method of citrus
pests (QCVN-01-119:2012/ BNNPTNT) and Pham Chi Thanh [11].
2.3.2. Methods for sample collection and isolation
2.3.2.1. Describe the symptoms of jackfruit trunk canker
Both external and internal tissue symptoms of jackfruit trunk
canker as well as root symptoms were observed and described.
2.3.2.2. Sample collection method
Following the method described by Drenth and Sendall (2001),
symptomatic samples were collected from the tissue closed to the typical
lesions on the infected roots, stems, leaves and fruits. Soil and root samples
were collected from the 20 x 20 x 20 cm holes that located at the edge of
the canopy shadow, but not the 5 cm-topsoil.
2.3.2.3. Isolation method


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- Direct isolation from jackfruit tissue: the pieces of jackfruit tissue
were placed onto Petri dishes that containing common media (WA, CRA
and PDA) or selective media [P10ARP, P5ARP and P5ARP(H)] at 25 ± 1oC
in the dark to isolate fungus pathogen.
- Phytophthora baiting using rose petals (Rosa sp.) (Drenth and
Sendall (2001), Nguyen Van Tuat and Pham Ngoc Dung (2012). Total 73
samples (35 root, 28 trunk, 6 leaf and 4 fruit samples) were collected. From

range from 9oC to 36oC with the 3o interval. The number of sporangia was
recorded at 48 hours after sporulation stimulation. Each treatment was
repeated 3 times, 3 Petri dishes each time.
2.3.4.3. Study the effect of pH
MD5 was inoculated on PDA at pH from 4 to 9 with the 0.5
interval. The growth of the colony diameter was calculated based on the
difference between the 5th day and the 2nd day after inoculation.
2.3.4.4. Testing the resistance to Metalaxyl of the Phytophthora isolates
The floating leaf-disc assay was used to test the resistance of 10
Phytophthora isolates (MD1 to MD10) to Metalaxyl following RunnoPaurson et al. (2016). Six leaf discs (14 mm) were floated in 90 mm Petri
dishes, each containing 10 ml of Metalaxyl solution at 0; 10 and 100
mg/liter for 3 treatments (4 replicates,1 Petri dish/replicate). Each leaf discs
were inoculated with 20 µl dilution at 104 sporangia/ml. After inoculation,
the discs were incubated at 27 ± 1°C. If sporangia were formed on 100
mg/liter Metalaxyl leaf discs: resistant isolate, on 10 mg/liter discs: tolerant
isolate, and on 0 mg/liter discs: susceptible isolate.
2.3.4.5. Pathogenicity test
The floating leaf-disc assay (Hermansen et al., 2000) was used to
test with the leaves of breadfruit (A. altilis), To Nu (A. integer) and durian
(Durio zibethinus). Six of 15-mm-diameter leaf discs were inoculated with
20 µl each of Phytophthora MD5 spore at 105/ml at 27°C on wet Whatman
paper in a Petri dish. Each plant species had 4 replicates, 1 Petri


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dish/replicate, six leaf discs/dish. Check leaf discs for any lesion, hyphae,
or spore formed at 7 days after inoculation under microscope.
2.3.5. The epidemiology of the jackfruit canker in the field
2.3.5.1. Survey of disease incidence and severity in the fields

Sieu Som orchards at productive stage, having >800 trees/orchard,
stretching from the foothills to the top of the hill with a slope from 4-16%.
Each orchard was divided into 3 areas (>200 trees/are) across slope
direction, including the lowest area (called the foothill), the middle area
(called the side of the hill) and the highest area (called the top of the hill).
e) The effect of inter-cropping
The disease incidences (%) were investigated in monocrop
jackfruit orchards and inter-cropped jackfruit orchards with pineapple
(Ananas comosus), or cashew (Anacardium occidentale), or durian (Durio
zibethinus). Each orchard type had 5 orchards (Sieu Som, > 3 years old).
2.3.6. Study the management for jackfruit canker
2.3.6.1. Screening tolerant jackfruit variety to P. palmivora
a) Evaluating the tolerance of jackfruit varieties to P. palmivora using
detached leaf and young stem bioassay
Detached leaf and stem bioassays were conducted from Jan. to
Dec. 2015 in lab condition following Sangchote, 2002 and O’Gara et al.,
2004. P. palmivora MD5 was inoculated for 22 varieties including Khong
Hat, La Lon (La Bang), M102, M97I, M98, M99, Ma Lai, MBRVT32H,
MBRVT33H, MDN02H, MDN06H, MDN07H, MDN09, MTNDN04,
MTNDN05, MTNDN06, MTNDN07, MTNDN08, Ruot Đo, Sieu Som, To
Tay and Vien Linh for screening the tolerant varieties against P. palmivora
b) Evaluating the tolerance of seedlings planted from seeds of tolerant
jackfruit varieties
Seedlings of 7 varieties including Ma Lai, MTNDN04, MTNDN05,
MTNDN06, MTNDN07, La Lon and Sieu Som were evaluated the
tolerance against P. palmivora from 1/2015 to 12/2016 in the roofed net
house. Uniform seedlings in pots filled with pasteurization potting mix
reaching 9-10 leaves were chosen to set up on the bench (50 cm above





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Phytophthora MD5 was used as inoculum. Disease incidences and severity
(%) were recorded at 12 months after inoculation.
d) Effect of organic manure on trunk canker under field condition
The ability of chicken and cow manure to reduce jackfruit trunk
canker epidemic was evaluated in Sieu Som orchard (6 x 5 m spacing, 4
years old, no manure amendment before, no fungicide application) in
xanthic ferralsols soil in Loc Ninh - Binh Phuoc province. The RCBD
experiment had 5 treatments: i) 12 ton/ha chicken manure; ii) 6 ton/ha
chicken manure; iii) 16 ton/ha cow manure; iv) 8 ton/ha cow manure; v) no
manure (control); 4 replicates, 16 seedlings/replicate. Disease incidences
(%), disease severity (%) and yield (kg/tree/year) were recorded.
2.3.7. Effect of T. harzianum on trunk canker under field condition
T. harzianum SR18 (108 spore/g product, 30 g/1 m canopy) were
dissolved in water at 2.5 g/l before spraying or drenching for Sieu Som
orchard (4 x 4 m spacing, 4 years old) in Loc Ninh (Binh Phuoc) from
5/2015-5/2017. The RCBD experiment had 6 treatments: i) sprayed T.
harzianum SR18 3 times (May, Jul., Sep.); ii) sprayed T. harzianum SR18
6 times (monthly from May-Oct.); iii) drenched T. harzianum SR18 2 times
(Jun., Aug.); iv) drenched T. harzianum SR18 3 times (May, Jul., Sep.); v)
drenched T. harzianum SR18 3 times (May, Jul., Sep.) + sprayed T.
harzianum SR18 3 times (Jun., Aug., Oct.) and vi) sprayed and drenched
water (control); 4 replicates, 15 plants/replicate. Disease incidences (%),
severity (%) and yield (kg/tree/year) were recorded.
2.3.8. Study the chemical measure for controlling trunk canker
a) Efficacy of fungicides on trunk canker in nursery condition
Sieu Som plants that infected with P. palmivora in the potting mix

The model was conducted in 3 Sieu Som jackfruit orchards from
1/2014 – 12/2016. Each orchard was divided into 2 plots: the model plot (for
the integrated management practices) and the control plot (disease
management following farmer’s practices). The first orchard was 4 years old,
spacings 7 x 7 m in Xuan Loc (Dong Nai) with the 3,000 m2 model plot and


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the 4,500 m2 control. The second orchard was 5 year-old, Sieu Som; 7 x 7 m
spacings in Dong Xoai (Binh Phuoc) with the 3,600 m2 model plot and the
3,700 m2 control plot. The third orchard was a 5 year-old, Sieu Som; 7 x 7 m
spacings in Loc Ninh (Binh Phuoc) with the 5,000 m2 model plot and the 6,000
m2 control. The integrated managements in 3 model plots were: i) Orchard
sanitation; ii) Building drainage system with 30 cm deep x 30 cm wide ditches
between rows; iii) Amending 12 ton/ha/year chicken manure in May; iv)
Drenching 1% T. harzianum SR18 in May, Sep. (1 g product for 1 m
canopy diameter) and spraying 3g/l in Jun., Aug., Oct. (2 g product for 1 m
canopy diameter); v) Canopy spraying 1% P. phosphite 200 g/l; 6.25
l/ha/time in May, Aug. and drenching in Jul., Sep. Disease incidences (%),
corrected efficacy using Henderson-Tilton’s formula, fruit weight
(kg/fruit), yield (kg/tree/year) and economic efficiency were recorded.
2.3.10. Data process
Data was statistically analyzed by SPSS program, version 22.0.
The data was appropriately transformed according to statistic rules before
processing if necessary. The productivity averages of each integrated
management model were statistically analyzed by t-test.
Chapter III: RESULTS AND DISCUSSION
3.1 The symptoms of jackfruit trunk canker in the Southeastern region
The initial symptom was a small discoloration spot on the bark,

Figure 3.3. Leaf blight symptom

Figure 3.4. Fruit rot symptom


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3.2. Isolation and identification the causal agents
- Isolation from infected samples: The 49 isolated fungus samples
were initially supposed to be Phytophthora species based on morphological
characterization. Isolated Phytophthora (MD5 and MD6) was re-inoculated
into healthy jackfruit seedling (on the trunk), healthy leaves and healthy
fruits that showed the same typical symptoms as originally observed.
Phytophthora was also re-isolated and shown to be identical to the
originally inoculation. Thus, Koch’s postulate (1876) was fulfilled.
- Identification of Phytophthora species causing jackfruit trunk
canker: based on the morphological classification key by Ho (1992) [74],
10 isolates MD1-MD10 were identified as P. palmivora (E.J. Butler) that
was described [56], [59], [131]. The analysis of the COX II sequences of 4
representative isolates (MD3, MD5, MD6 and MD8) revealed 99%
similarity with several reference isolates of P. palmivora. The ITS1-5.8S ITS2 sequences of these 4 isolates also showed the similarity with the
reference sequences of P. palmivora. Therefore, P. palmivora was affirmed
to be the causal agent for jackfruit trunk canker in the Southeastern region.
3.3. Study on biological characteristics of P. palmivora isolates
All ten isolates were heterothallic and were of A1 mating type,
producing amphigynous antheridia, oogonia, and oospores after pairing
with the A2 reference isolates of P. nicotianae. Minimum and maximum
temperatures for growth on PDA were, respectively, 9°C and 36°C and the
optimum temperature for growth and sporangial production was 27oC. The
optimum pH range for growth was between 5 and 7. P. palmivora MD5

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The disease incidence (%) of the Sieu Som was the highest,
16.82% in 2013; 21.84% in 2014 and up to 25.63% in 2015; followed by
Vien Linh (with the incidence of 10.32%, 13.73% and 20.35%
respectively). Among common jackfruit varieties, the La Lon had the
lowest incidence; corresponding to 2.71%; 4.75% and 4.36%. Most
varieties had disease incidence developed from 2013 to 20159 (Table 3.9).
3.5.2. The effect of planting spacings
Planting spacing at 7 x 7 m had the lowest disease incidence (2013:
13.23% and 2014: 15.20%). The smaller the planting spaces were (5 x 5 m;
4 x 4 m and 3 x 3 m), the higher the disease incidence were (corresponding
to 17.25%; 21.76% and 24.48% in 2014). Thus, the jackfruit orchards with
small planting spaces (3 x 3 m; 4 x 4 m) had higher disease incidence than
the ones with appropriate planting density (7 x 7 m) (Table 3.10).
3.5.3. The effect of tree ages
In year 2014, the orchards with 1-2 year-old trees had the lowest
disease incidence (8.61%), following by the orchards with 3-4 years old
trees (15.90%); 5-6 years old trees (22.60%) and the ones > 6 years old
trees (25.80%). Therefore, the plant age affected the disease incidence; the
older orchards (> 6 year-old trees), the more severe canker (Table 3.11).
3.5.4. The effect of different topography
The orchard topography affected the incidence (%). The 2014
survey showed that the orchards at low area (foothills) had higher disease
incidence (25.50%) than the ones at middle area (the side of the hill,
17.42%) and the ones at high area (top of the hill, 14.52%). The orchards
on flat land (with slope
The percentage of symptomatic plants was 0.11-0.19 % averagely
in surveyed nurseries. P. palmivora was detected in the nurseries (8090.9%). Therefore, infected nursery plants could be a pathway for the
dispersal of strunk canker (Table 3.17).


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3.6.2.2. Analyze the effect of drainage methods on jackfruit trunk canker
Three years after treatments, the treatment of making pool with
high edge around each tree for hose irrigation had the highest disease
incidence (21.64%) and severity (9.16%); following by the treatment of no
mound and no ditches (18.34% and 7.50% respectively). The disease
incidences of these two treatments were statistically significant higher than
the others. Thus, mounding and ditching were found efficiently to prevent
disease outbreak and reduce disease epidemic (Table 3.18; 3.19).
3.6.2.3. The effect of manure on trunk canker
a) The effect of manure on trunk canker in jackfruit in pots
The disease incidence was lowest in the chicken manure treatment
(11.67%), following cow manure (20.00%); significantly lower than the
infection treatment with inorganic fertilizer (49.17%) and the infection
treatment without fertilizer (55.00%). The chicken manure treatment had
the lowest percentage of P. palmivora re-isolation (8.34%), following cow
manure treatment (11.67%), significantly lower than the other two.
Therefore, applications of chicken and cow manure could reduce the
disease incidence of the jackfruit seedlings in nurseries (Table 3.21-3.23).
b) The effect of manure on jackfruit trunk canker in field condition
The disease incidences among treatments were not statistically
significantly different 3 years after the treatments. However, disease
severity (%) were statistically different; 12 ton/ha chicken manure
treatment had the lowest disease severity (5.31%), following by 16 ton/ha


Before

After

app.

app.

app.

app.

1 Spray 3 times

5,00

11,67b

2,00

7,00b

2 Spray 5 times

3,34

6,67bc

1,34


2,34d

6 No treatment (control)

6,67

23,34a

2,34

9,33a

F

ns

*

ns

*

CV (%)

25,38

14,38

27,12

Table 3.31. Efficacy of application methods of Potassium
phosphite to the jackfruit trunk canker under filed condition
Incidence (%)
No

Treatment

After Before

After

app.

app.

app.

5,56

12,50b

1,94

5,28b

2 Spray P. phosphite 1%

5,56

9,72bc


1,67

12,73a

F

ns

*

ns

*

CV (%)

23,27

11,46

17,55

12,28

1 Spray Metalaxyl M 40 g/l +
Mancozeb 640 g/l; 0,3%

Before


biological products and labor. Howerver, average yield and fruit price of
the models were higher than control (16.94% -equivalent to 7.79 tons/ha
and 15.69% higher respectively), thus its profits were higher than the
control. The profit rate of the models (6.76) was higher than the controls
(5.55). The difference between the models and the controls was 21.74%.


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CONCLUSION AND SUGGESTION/RECOMMENDATION
4.1. Conclusion
- Based on the morphological and biological characteristics, the
results of re-inoculation and re-isolation (fulfilling Koch’s postulates) as
well as on the ITS sequences, P. palmivora was identified as the causal
agent of the jackfruit trunk canker in the Southeastern region. P. palmivora
was also firstly found causing trunk canker on jackfruit in Vietnam.
-The optimum temperature for P. palmivora growth and sporangial
production was 27oC, and the optimum pH range for growth was between 5
and 7.
- Jackfruit trunk canker was widespread in rainy season (May- Oct.).
The disease epidemic rose up starting from May (as precipitation fell often)
to July and continued to increase and to keep up at severe level until Oct.
(at the end of the rainy season). From Dec., the disease incidence was
dropped up to very low level (almost negligible from Feb to Apr.).
- The field survey revealed that the disease epidemic was found
severe in the poor drainage or high planting density orchards. The Sieu
Som orchards had higher disease incidence than the La Lon orchards. The
intercropped orchards of jackfruit and pineapple or durian had higher
diseases incidence than monoculture jackfruit orchards.
- La Lon was evaluated as the most tolerant variety whereas the Sieu