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 MA
Trang 1AND 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
Trang 2.TS NGUYỄN VĂN TUẤT
This thesis was completed at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
Supervisors: Prof Dr Nguyen Van Tuat
Dr Nguyen Van Hoa
This thesis can be referred at:
1) The National library
2) The library of Vietnam Academy of Agricultural Sciences
3) The library of Institute of Agricultural Science for Southern Vietnam
Trang 31.1 Rationale of research
Jackfruit (Artocarpus heterophyllus Lam.; Moraceae) is currently one
of the high value crops in Vietnam with more than 24,000 ha The Southeastern is as the top two producing and processing regions According
to the Department of Crop Production, jackfruit export value in 2017 was estimated at 28 million USD, higher than that of rambutan Due to high demand, jackfruit growing areas have been expanded rapidly in the region Trunk canker, a serious disease, has been causing significant losses for jackfruit production [5] Trunk canker affects tree growth and crop production, reducing productivity and longevity of jackfruit, thus trunk
canker is one of the principal limiting factors in jackfruit production in the
Southeastern region Therefore, the study on the disease causal agent and control measures are crucial to build up the management strategies of the trunk canker and the survival of the jackfruit industry
1.2 Purpose of the study
Identifying the causal agent of the jackfruit trunk canker and studying measures for controlling the disease in the Southeastern region
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
Trang 4thesis 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: (1) Using tolerant variety La Lon; (2) Building ditches between rows and planting on raised mound for good drainage in the orchards; (3) Amending chicken manure (12 tons/ha/year) or cow manure (16 tons/ha/year); (4) Soil
drenching twice combined with canopy-spraying Trichoderma harzianum
SR18 three times during rainy seasons (May-Oct.); and (5) Canopy spraying twice alternating with soil drenching twice Potassium phosphite 1% during rainy seasons
1.5 Structure of the thesis
The thesis had 208 A4 pages including 36 tables, 20 figures and Annexes that were listed in order as: Introduction (4 pages); Chapter I: Literature review (38 pages); Chapter II: Materials and Methodologies (35 pages); Chapter III: Results and Discussion (84 pages); Conclusion and Recommendations (2 pages)
Trang 5Chapter I LITERATURE REVIEW
Jackfruit trunk canker disease (also known as foot rot-gummosis, trunk splitting-gummosis and other names) has been presented in Vietnam
for a long time and was suspected to be caused by Phytophthora [2], [3]
Symptoms of jackfruit trunk canker were characterized by a reddish brown discoloration patch of the trunk outer bark, areas of necrosis on the bark, and a reddish brown discoloration in the outer sapwood The disease also affects on root, leaf and fruit causing root rot, blight leaf and fruit rot
Phytophthora species, including P palmivora, have been associated
with fruit rot, stem canker, and root rot of the related species as breadfruit
(A integer) and cempedek (A altilis) [50], [124] Phytophthora has not
previously been confirmed as the cause of trunk canker disease on jackfruit
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
Trang 6important 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 control measures, and the judicious application of appropriate chemicals [47] The principle of integrated management of Phytophthora diseases in durian has been promoted since 2004 [47] A Phytophthora management program on durian developed including five measure groups based on (1) cultivation, (2) resistance/tolerance, (3) biological control, (4) fungicides, and (5) Phosphonates application [47] This systematic approach will be consulted for the development of integrated control strategies for Phytophthora diseases on jackfruit in the Southeastern region
Chapter II: MATERIALS AND METHODOLOGIES
2.1 Materials
Jackfruit trees at different ages in farmers’ orchards that were propagated by grafting were used as materials for field research Jackfruit trees for experiments in net houses could be germinated from seeds or propagated by grafting depend on the purpose of study The fungus media were used in this thesis including the common media, such as WA, CMA,
V8A, CRA and PDA; the selection media for Phytophthora, such as
Trang 7P10ARP, P5ARP, P5ARP(H); mass production media and others The materials that were used in this thesis also included all essential chemicals, equipment and tools for isolating and identifying the pathogens as well as for microbiology and molecular studies (made in China and Germany); fungicides and fertilizers (domestic goods); and other disease control products (both domestic and imported)
2.2 Research duration and location
This study was conducted from Sep 2012 to Mar 2018 at the Southern Horticultural Research Institute (SOFRI) in Tien Giang and the Southeast Horticultural Research Center (SeHort) in Ba Ria Vung Tau (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
Trang 8- 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 these samples, 49 isolates were successfully isolated, then 10 isolates were randomly chosen and named from MD1 to MD10 for further experiments
2.3.3 Identification methods for the disease causal agent
2.3.3.1 Morphological identification of Phytophthora species
The Phytophthora isolates (MD1 to MD10) were examined
morphologically for species identification based on the keys by Stamps et
al (1990), Erwin and Riberrio (1996) and Gallegly and Hong (2008)
2.3.3.2 Molecular identification of Phytophthora species
Four Phytophthora isolates (MD3, MD5, MD6 and MD8) were
sequenced DNA of these isolates were extracted by 2 different methods and sequenced at 2 different DNA regions The identity of these isolates was found based on the level of similarity (using the BLAST tool) of their
COX II and ITS sequences with those of reliable reference sequences of Phytophthora species that were recorded in the GenBank (NCBI)
2.3.4 Study on biological and ecological characteristics
These experiments were conducted from Jan to Dec., 2013 at the Biotechnology and Plant Protection Labs of SeHort
2.3.4.1 Determination of mating type of the Phytophthora spp
The 10 isolates (MD1 to MD10) were used with known tester A1
(P palmivora) or A2 (P nicotianae) on V8A (Brasier et al., 2003) If the
isolates that mating with A1 formed oospores, the isolates were A2 and vice versa, the isolates were A1 if it formed oospores with A2
Trang 92.3.4.2 Study the effect of temperature
C-39oC with the 3o interval on CRA medium in the dark The mycelium diameter was recorded at 2; 4 and 6 hours after inoculation The effect of temperature on the MD5 sporangial production was also investigated at 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
Runno-Paurson 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
Trang 10dish/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
The survey of disease incidence and severity were conducted monthly in the fields in 2 years 1/2013-12/2014 Rainfall data were also collected monthly The method of survey was mentioned in 2.3.1
2.3.5.2 Study the effects of ecological factors on the disease incidence
Sampling points were on the two diagonal lines and other 5 points
on the representative crop rows of the orchards Each point, 5-20 trees were investigated to determine the disease incidence (%) Each orchard, 5-20 root and soil samples were collected to initially detect pathogen using baiting method as mentioned above
a) The effect of different jackfruit varieties
The disease incidences (%) of 10 orchards (productive stage, >3 years old) for each commonly cultivated jackfruit variety were investigated
b) The effect of planting spacing
The disease incidences (%) were investigated in the Sieu Som orchards with various planting spacing 7 x 7 m; 5 x 5 m; 4 x 4 m and 3 x 3 m; each spacing, 10 orchards (≥200 trees/orchard) at the productive stage
c) The effect of tree ages
The disease incidences (%) were investigated in the Sieu Som jackfruit orchards at different age stage from 1-2 years, 3- 4 years, 5- 6 years and > 6 years; 10 orchards (≥200 trees/orchard) per age stage
d) The effect of different topography
The disease incidences (%) were investigated in the Sieu Som jackfruit orchards on different topography including flat, slightly sloping (<3%) and sloping land (4-16%) [135]; 10 orchards (≥200 trees/orchard) at productive stage (> 3 years old) for each topography type
Trang 11The disease incidences (%) were also investigated at another 10 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
Trang 12ground) for inoculation with Phytophthora MD5 This RCBD experiment
had 7 treatments (7 varieties with Sieu Som as a control), 4 replicates, 14 seedlings/replicate The disease incidences (%) were recorded
2.3.6.2 Effect of cultural measure for controlling jackfruit trunk canker
a) Investigating the P palmivora prevalence on jackfruit nursery plants
The disease incidences (%) and P palmivora prevalences (%) were
investigated from the nurseries in BRVT (6), in Dong Nai (15), in Binh Duong (5) and in Binh Phuoc (11) from 1/2016-12/2016 on Sieu Som
variety The infected nursery plants and prevalence of P palmivora (%) on
pot mix were determined from 30-50 randomly seedling samples/nursery
b) Effect of drainage practices on jackfruit trunk canker
The effects of drainage methods on disease incidence (%) and severity (%) were evaluated in Sieu Som orchards (3 years old, 4 x 4 m spacing) in Loc Ninh (Binh Phuoc), from 1/2013-12/2015 The RCBD experiment had 4 treatments based on the farmers’ convetional drainage practices: i) Building irrigation pool with high edge around trunk (for hose irrigation); ii) No irrigation pool + building drainage ditches; iii) Making mound and drainage ditches; iv) No mound and ditches (control) Each treatment had 5 replicates and 24 trees/replicate
c) Effect of organic manure on trunk canker of potted jackfruit plants
One-month Sieu Som seedlings from the same mother tree were potted in 20 x 20 cm containers filled with pasteurization potting mix The
RCBD experiment had 5 treatments : i) Uninoculated Phytophthora + no manure (control); ii) Inoculated Phytophthora + no manure; iii) Inoculated
Phytophthora + no manure but applied 3 times of NPK 20-20-15 (in the
potting mix, 1 and 3 months after planting, 10 g/pot each time); iv)
Inoculated Phytophthora + chicken manure (300 ml); v) Inoculated
Phytophthora + cow manure 500 (ml); 4 replicates, 15 seedlings/replicate
Trang 13Phytophthora 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
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
from selected nurseries were used The RCBD experiment in the roofed net house had 5 treatments: i) Copper oxychloride 850g/kg WP (0.25%); ii) P phosphite 200 g/l (1 %); iii) Fosetyl -Al 800 g/kg WG (0.25%); iv)