Nghiên cứu tác nhân gây bệnh và biện pháp phòng trừ bệnh thối thân chảy nhựa trên cây mít ở miền đông nam bộ tt tiếng anh
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
.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: ……………………………….... Reviewer 3: ………………………………….
The dissertation would be defended against the Dissertation Committee of Vietnam Academy of Agricultural Sciences at Institute of Agricultural Science for Southern Vietnam, ………………….
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
INTRODUCTION 1.1. 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 . 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
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: (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).
Chapter 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 , . 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) , . 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 . Many species can be easily identified by morphological techniques using a number of morphological and physiological characteristics that were typically classified by Waterhouse (1963) , Stamps et al (1990)  and Ho (1992) . 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 . Therefore, it is
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 . Effective disease control is rarely achieved through the application of a single control measure  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 . The principle of integrated management of Phytophthora diseases in durian has been promoted since 2004 . 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 . 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
P10ARP, 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 . 2.3.2. Methods for sample collection and isolation 126.96.36.199. 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. 188.8.131.52. 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. 184.108.40.206. Isolation method
- 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 220.127.116.11. 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). 18.104.22.168. 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. 22.214.171.124. 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.
126.96.36.199. Study the effect of temperature Phytophthora MD5 was tested at a temperature range from 3oC39oC 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. 188.8.131.52. 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. 184.108.40.206. 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. 220.127.116.11. 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
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 18.104.22.168. 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. 22.214.171.124. 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%) ; 10 orchards (≥200 trees/orchard) at productive stage (> 3 years old) for each topography type.
The 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 126.96.36.199. 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
ground) 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. 188.8.131.52. 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.
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 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)
Metalaxyl M 40 g/l + Mancozeb 640 g/l (0.3%); v) water spray (control); 5 replicates, 35 plants/replicates. The disease incidence (%) and P. palmivora prevalence (%) in potting mix were recorded. b) Efficacy of fungicides on trunk canker under field condition The Sieu Som orchard (3 x 3 m spacings, 4 years old) in Dong Xoai (Binh Phuoc) was tested from 1/2013-2/2014. The RCBD experiment had 5 treatments: i) Potassium phosphite 200 g/l (0.5 %); ii) Copper oxychloride 850g/kg WP (0.3%); iii) Fosetyl -Al 800 g/kg WG (0.25%); iv) Metalaxyl M 40 g/l + Mancozeb 640 g/l (0.3%); v) water spray (control); 4 replicates, 18 plants/replicate. All sprays were a 30-day interval from May to October. Disease incidences (%), disease severity (%) and fruit yield (kg/tree/year) were recorded. c) Efficacy of various application of P. phosphite on trunk canker The RCBD experiment had 4 replicates, 18 plants/replicate, 5 treatments: i) sprayed canopies with P. phosphite 200 g/l (1 %) (4 times, 6week interval, started at the ending May); ii) drenched with P. phosphite 4 times; iii) sprayed canopies twice and drenched twice with P. phosphite; iv) sprayed 4 times with Metalaxyl M 40 g/l + Mancozeb 640 g/l (0.3%); v) sprayed and drenched clean water as control. No other fungicide was applied in the experimental Sieu Som orchard (4 x 4 m spacings, 4 years old) in Loc Ninh (Binh Phuoc) from 1/2015 to 12/2016. Disease incidences (%), disease severity (%) and yield (kg/tree/year) were recorded. 2.3.9. Development the integrated management model to control jackfruit trunk canker 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
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, then the lesion expanded and water soaked, and finally resins were exuded through the lesion. The wood tissue inside the lesion was dark brown rot steadily spread through the vascular bundles (Figure 3.1). The widely spread lesion would turn into the trunk crack. The disease progress was normally slow with various symptoms such as poor growth, leaf yellowing, deciduous, dye back, decline and gradual death in a few months to several
years. The susceptible varieties died faster than other varieties. Infected trees gradually reduced their growth and productivity with small fruits. The infection on root system caused root rot; on leaves caused burning leaves; and on fruits caused fruit rot (Figure 3.2, Figure 3.3 and Figure 3.4). Symptoms above ground occurred only in the conditions of high humidity and continuously gloomy days.
Figure 3.1. Symptom of jackfruit trunk canker: Cracking dark brown lesion (a and b)
Figure 3.2. Root rot symptom on young (a) and mature (b) roots
Figure 3.3. Leaf blight symptom
Figure 3.4. Fruit rot symptom
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) , 10 isolates MD1-MD10 were identified as P. palmivora (E.J. Butler) that was described , , . 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 could infect on breadfruit (A. altilis), cempedak ‘To Nu’ (A. integer) và durian (D. zibethinus). All 10 Phytophthora isolates (MD1 to MD10) were found susceptible to Metalaxyl. 3.4. The epidemiology of the jackfruit canker under field condition 3.4.1. The disease occurrence under field conditions
The disease incidences of 4 investigated provinces in the Southeastern were from 14.5 - 34% (34% of 1,962 trees in Dong Nai, 20.5% of 1,892 trees in Binh Phuoc, 19% of 2,126 trees in BRVT and 14.5% of 1,540 trees in Binh Duong). 3.4.2. The disease progress during years In the dry season of 2013 (from Dec. to Apr.), the percentage of newly infected trees per month was very slow (0-0.1%). From May, as the rainfall increased gradually, the disease occurence began to increase gradually and reached the peak in Oct. (2.2%). After that, the percentage of newly infected trees per month gradually decreased to 1.6% in Nov. and then to 0.1% in Dec. (Figure 3.15). The result in year of 2014 was similar to 2013. Thus, trunk canker disease occured severely in the rainy season (May-Oct) with delayed period of one month and at a very low rate in the dry season (from Nov. to Apr.).
Figure 3.15. The percentage of newly infected trees (%) per month and the average monthly rainfall during 2013 in the Southeastern region 3.5. Study the effects of some ecological factors on the disease incidence 3.5.1. The effect of different jackfruit varieties
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 <2%) had higher disease incidence, 21.81%, than the ones on sloping land (with slope 10-30%), 15.80%. (Table 3.12) 3.5.5. The effect of inter-cropping From the results in 2014, the disease incidence (%) of the orchards intercropped with pineapple (27.36%) and durian (26.53%) were higher
than those of the monoculture orchards (17.64%). Therefore, it is suggested that should not to intercrop jackfruit with pineapple and durian (Table 3.13) 3.6. Study the management for jackfruit trunk canker 3.6.1. Screening jackfruit varieties for the tolerant to P. palmivora 184.108.40.206 Evaluating several jackfruit clones for the tolerant to P. palmivora under net-house The 22 jackfruit varieties were categorized into 3 tolerant groups: i) Tolerant varieties (La Lon, Ma Lai, To Tay, MTNDN04, MTNDN05, MTNDN06, MTNDN07 and MTNDN08), in which La Lon had the highest tolerance, ii) Highly susceptible varieties (Sieu Som, Ruot Do, Vien Linh, M97I), in which Sieu Som was the most susceptible variety; iii) Susceptible variety (intermediate group: Khong Hat, M102, M98, M99, MBRVT32H, MBRVT33H, MDN02H, MDN06H, MDN07H, MDN09H). This result was also consistent with the results from the field survey. Sieu Som was highly susceptible while La Lon was tolerant to P. palmivora (Table 3-14; 3.15). 220.127.116.11. Evaluating the tolerance of potted seedlings planted form seeds from tolerant jackfruit varieties to P. palmivora. Young seedlings of 7 investigated varieties were all infected by the inoculum P. palmivora in the pots. Two months after inoculation, disease incidence (%) of the Sieu Som seedlings was the highest (55.72%) that statistically significantly higher than the others. In contrast, the La Lon seedlings had the lowest disease incidence (27.14%) (Table 3.16) 3.6.2. Jackfruit canker management by cultural practices 18.104.22.168. Investigate the P. palmivora prevalence in jackfruit seedlings in nurseries 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).
22.214.171.124. 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). 126.96.36.199. 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 cow manure treatment (7.5%); significantly lower than control (11.25%). Average fruit yield (kg/tree/year) over three years varied from 32.85 to 51.21 kg/tree. Applications of high amount of chicken manure (12 tons/ha) and cow manure (16 tons/ha) increased fruit yield compared to control. (Table 3.24-3.25)
3.6.3. Jackfruit trunk canker management by application of T. harzianum in field condition In the second year after application, the treatment of drench twice + 3 times spraying of T. harzianum SR18 in rainy seasons had the lowest disease incidence and severity (5.00% and 2.34% respectively); following by the treatment of 5 times spraying/year (6.67% and 4.00%) (Table 3.24). Fruit yield (kg/tree/year) was highest in the drench twice + 3 times spraying treatment (51.05 kg), following by 5 times spraying treatment (48.95 kg) Table 3.24. The effect of T. harzianum SR18 on the jackfruit trunk canker incidence (%) and severity (%) at productive stage No
T. harzianum SR18
1 Spray 3 times
2 Spray 5 times
3 Drench twice
4 Drench 3 times
5 Drench twice + spray 3 times
6 No treatment (control)
3.6.4. Study the chemical control for jackfruit trunk canker 188.8.131.52. The efficiency of several chemicals to control jackfruit trunk canker in nursery condition Fosetyl-Al spraying treatment had the lowest disease incidence (3.43%), followed by Potassium phosphite spraying (5.71%), lower than the control (13.72%). P. palmivora prevalence (%) of the treatment of Fosetyl-Al (66.89%) and P. phosphite spray (63.43%) lower than the
control (78.86%). Thus, Fosetyl-AL followed by P. phosphite had the highest control efficacy in nurseries. 184.108.40.206. The efficacy of spraying chemicals to control jackfruit trunk canker at productive stage in field condition Over two years of the treatment, the lowest disease incidence (%) was recorded in Fosetyl-Al spraying treatment (6.95%) and P. phosphite treatment (8.34%). Similarly, disease severity (%) of the Fosetyl-Al treatment (2.50%) and the P. phosphite treatment (3.89%) were the lowest. Fruit yield (kg/tree/year) was highest from the P. phosphite treatment (45.94 kg), followed by the Fosetyl -Al 800g/kg treatment (40.06 kg). Thus, all of the 4 chemicals used in this experiment reduced the disease incidences (%) and severity (%) compared to the control; in which Fosetyl Al and P. phosphite had the highest efficacy. 220.127.116.11. The efficacy of various application methods of Potassium phosphite to control jackfruit trunk canker at productive stage Table 3.31. Efficacy of application methods of Potassium phosphite to the jackfruit trunk canker under filed condition Incidence (%) No
In the second year, the incidences of treatments by spraying + drenching (5.56%), by drenching treatment (6.95%) and by spraying + drenching (2.50%) of P. phosphite were lower than those of Metalaxyl M 40 g/l + Mancozeb 640 g/l. (Table 3.31). Fruit yield (kg/tree/year) reached the highest in both treatments, spraying + drenching with P. phosphite (49.52 kg/tree/year) and only drenching P. phosphite (48.46 kg/tree/year), that significantly higher than controls (31.86 kg/tree/year) and than the two other chemical treatments. Therefore, Metalaxyl application might be partially replaced by P. phosphite for jackfruit trunk canker management to ensure alternative chemical applications. In addition, P. phosphite would be useful as more options for the disease control and satisfied the regulations of chemical residues for various markets. 3.6.5. The integrated management model for controlling jackfruit trunk canker The average disease incidences (%) of the management models were lower than the controls. The efficacy of disease control of the models was ≥ 90%, of which, the first model was 92.10%; the second model reached 93.86% and the third model was 90.00%. The average yield over 2 years implementing at the first model was > 31.71 kg/tree, at the second model was more > 41.23 kg/tree and at the third model was > 41.66 kg/tree/year compared to the controls. The averarhe cost of the models was 81.80 million VND/ha that was higher (14.25%) than control due to increasing investment in manure, 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%.
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 Som was the most susceptible variety against P. palmivora by detached leaf and young stem bioassays as well as field surveys. - The seedlings from nurseries could be the great potential inoculum because about 80% of the investigated nurseries were infested with the P. palmivora prevalence in potting mix from 7.04-17.16%.