BIOLOGICAL CONTROL OF CITRUS ROOT ROT IN CAMBODIA

Transcription

1 BIOLOGICAL CONTROL OF CITRUS ROOT ROT IN CAMBODIA Imperial Mae Ping Hotel, Chiang Mai June 2016 KEAN SOPHEA

2 Research 0utline 1. Collection, isolation, identification and Pathogenicity test. 2. Screening for antagonistic fungi. 3. Biological control of citrus root rot using biofungicides in field experiment

3 BACKGROUND Citrus sinensis is one of the most popular and also one of the economic crop in Cambodia Now, it has become a high demand in domestic markets The production is limited, due to pest, disease problems and lack of skills in farm management

4 SYMPTOM OF ROOT ROT Above the plant

5 SYMPTOM OF ROOT ROT On stem & root A B A = Infested field and dead plants, B = Gummosis symptom, C D C = Dead stem and main root D = Root rot

6 SYMPTOM OF ROOT ROT On fruit and post harvest

7 Objectives 1. To collect sample, isolate, identify and Pathogenicity test 2. To screen antagonistic fungi and study in laboratory 3. To evaluate the new antagonist against citrus root rot in field experiment

8 Objective To Collect, isolate, identify and Pathogenicity test Sample collection 140 soil samples were collected from infested field soil

9 Results Isolation and identification (baiting technique) Pythium ultimum: 16 isolates A. 3 day-old culture on PDA, B. mycelia, C. oospore, and D. Sporangium.

10 Results Pythium graminicola: 5 isolates A B C D A. 3 day-old culture on PDA, B. mycelia, C. oospore and D. Sporangium.

11 Results Pythium spinosis - 2 isolates A. 3 day-old culture on PDA, B. mycelia, C. oospore, and D. Sporangium.

12 2.3 Pathogenicity test Detached leave method CRD with 4 Rep Mycelia oospores germ tube infected leaf tissue The virulent isolates was determined according to disease index

13 Results Figure 2.1 Pathogenicity test of Pythium ultimum by detached leave method Control T 41 Control T 77 Control T85 Control T117 Control T128 5 isolates are the most virulence

14 Results Table 2. 1 Pathogenicity test of Pythium ultimum by detached leave method after 5 days Isolates Disease index (DI) 1 T a 2 T a T a T a T a Non-treated control 1.00b These 5 isolates were selected for pathogenicity test for root rot 1 Disease index (DI) of detached leave: level 1 = healthy or green leaves, level 2 = 1-25% yellow leaves, level 3 = 26-50% yellow leaves, level 4 = 51-75% yellow leaves and level 5 = % yellow leaves. 2 Average of four replications. Means followed by a common letter in the column were not significantly different (P=0.01) by Duncan s Multiple Range Test.

15 Pathogenicity test by root inoculation Zoospore suspension was adjusted to 10 6 zoospores/ml 30 days CRD with 4 Rep Disease severity index was recorded T41 T77 T85 T117 T128

16 Results Control T 41 Control T 77 Control T 85 Control T 117

17 Results Table Pathogenicity test of virulent isolates of Pythium ultimum by root inoculation Isolates Disease index (DI) 1 T41 4 a 2 T77 3b T85 3b T117 3b T b Control 1.c Root rot was proved to be caused by P. ultimum T41 which significantly higher disease severity than others. 1 Disease index of citrus root rot, level 1 = no root rot, level 2= 1-25% root rot, level 3=26-50% root rot, level 4=51-75% root rot and level 5=76-100% root rot (modified from Ahmed et al., 1999). 2 Average of four replications. Means followed by a common letter in the column were not significantly different (P=0.01) by Duncan s Multiple Range Test.

18 Conclusions Pythium graminicola: 5 isolates 5 Low virulent isolates Pythium spinosis: 2 isolates 2 Low virulent isolates Pythium ultimum: 16 isolates 5 high virulent isolates T 41, T 77, T 85, T 117 and T 128 The most virulent isolate - Pythium ultimum T 41

19 Conclusions Pathogenecity tests of detached leave and seedling test showed that Pythium ultimum T41 was proved to be seriously pathogen of citrus root rot in Battambang province. Pythium ultimum T41 was the most virulent causing root rot and followed by T77, T85, T117 and T128 which significantly different when compared to the non-treated one.

20 Objective To screen antagonistic fungi in laboratory Research Methodology Soil sample collection 60 soil samples from healthy citrus orchards Isolation and identification antagonistic fungi - Soil plate technique - Soil baiting technique

21 Results 34 isolates of promising antagonistic fungi 10 isolates- Soil plate technique 24 isolates- Baiting technique Aspergillus niger (1 isolate) Penicillium canescens (1 isolate) Trichoderma hamatum (1 isolate) T. harzianum (7 isolates) Chaetomium aureum (2 isolates) Ch. bostrychodes (4 isolates) Ch. globosum (7 isolates) Ch. indicum (3 isolates) Ch. cupreum (8 isolates)

22 Results This research finding is reported only the effective antagonists against Pythium ultimum and the ineffective antagonists were not reported and did not study further. Three effective antagonists against Pythium ultimum 1- Chaetomium globosum. 2- Chaetomium cupreum. 3- Trichoderma harzianum

23 Results A B C D Fig. 2.1 Chaetomium globosum. A. 10-day-old-culture on PDA, B. ascomata, C. ascomatal hairs, D. ascospores.

24 Results A B C C D D Fig. 2.2 Chaetomium cupreum. A. 10-day-old-culture on PDA, B. ascomata, C. ascomatal hairs, D. ascospores.

25 Results A B C DD Fig. 2.3 Trichoderma harzianum A. 7-day-old-culture on PDA, B.thalli, C. chlamydospores. D. conidia.

26 Research Methodology Dual Culture antagonistic test PDA Most virulent isolate (from pathogenicity test) Pathogen Bi-culture Antagonist colony diameter 1-30 days (data collection) Antagonistic fungus % colony inhibition < 50% - low antagonistic

27 Results Fig. 2.4 Bi-culture of Trichoderma harzianum BTB 022 vs. Pythium ultimum T41 5 days 7 days Percent of growth inhibition of Trichoderma harzianium BTB 022 is 60.33%

28 Results Fig. 2.5 Bi-culture of Chaetomium cupreum BTB 06 vs. Pythium ultimum T41 5 days 21 days Per cent of growth inhibition of Chaetomium cupreum BTB 06 is 70.88%.

29 Results Fig. 2.6 Bi-culture of Chaetomium globosum BTB 014 vs. Pythium ultimum T41 5 days 21 days Per cent growth inhibition of Chaetomium globosum BTB 014 is 66.44%

30 Results Table 2.3 Colony diameter (cm) and inhibition of pathogen in bi-culture antagonistic test against Pythium ultimum T41 Treatments C. globosum C. cupreum T.harzianum bi-culture 3.02 b b 3.75 b pathogen 9.00 a 9.00 a 9.00 a C.V.(%) Inhibition (%) The 3 effective antagonistic fungi could inhibit the colony of P. ultimum T41 with per cent of growth inhibition (PGI) 1 /Average of four replications. Means followed by a common letter were not significantly different (P=0.01) by DMRT.

31 Objective To evaluate the new antagonist against citrus root rot in field experiment Research Methodology The Integrated Pest Management (IPM) in combination of biological control agents was set up as a field experiment in the infested field planted to citrus at orchard of farmer approximately 0.75 hectares which was covered about 100 of 4 year-old citrus trees Experimental design The experiment was designed by using Randomized Completed Block Design with 4 replications. Location: Watkor village, Banann district, Battambang province, Cambodia The experiment was conducted in 1 year period

32 Research Methodology Experimental layout Total = 100 trees Rep I Rep II Rep III Rep IV T 1 T 2 T 3 T 4 T 5

33 Research Methodology Treatments T1= control was non-treated T2=chemical fungicide (metalaxyl) 20g/20 L of water spray at rhizosphere soil and above plants and applied chemical fertilizer 300 g/tree T3= chemical fungicide (metalaxyl) 20g/20 L of water and Chaetomium 10 g/tree at rhizosphere soil and above plants and applied chemical fertilizer 300 g/tree. T4= Chaetomium 20 g/20 L of water and applied 150 g/tree of chemical fertilizer and 5 kg/tree of compost. T5= Trichoderma 20 g/ 20 L of water and applied 150 g/tree of chemical fertilizer and 5 kg/tree of compost.

34 Research Methodology Experiment management Planting practices: spacing 4m x 4m Irrigation: the experimental plots were irrigated with stream water by pumping, particularly during the dry season Fertilization: monthly application

35 Research Methodology Experiment management Pesticide application: monthly application

36 Research Methodology Experiment management Weeding : this was carried out by monthly of the experimental period.

37 Research Methodology Experimental management Mulching

38 Research Methodology Data collection -Disease index was recorded as follows: -Level 1 = 0% of yellow or pale leaves, die back and root rot -Level 2 =1-20% of yellow or pale leaves, die back and root rot -Level 3= 21-40% of yellow or pale leaves, die back and root rot -Level 4= 41-60% of yellow or pale leaves, die back and root rot -Level 5= 61-80% of yellow or pale leaves, die back and root rot -Level 6:=81-100% of yellow or pale leaves, die back and root rot There were scored and evaluated by monthly during the whole experiment period.

39 Results Table Disease index after application of biological fungicides in the field. Disease index (months) Treatments control 2.91a 3.15a 3.36a 3.58a 3.71a 3.90a metalaxyl 2.92a 2.56ab 2.27a 1.96a 1.70b 1.46b GAP 2.81a 2.48a 2.18a 1.85a 1.56b 1.33b Ketomium 2.53a 2.25a 1.98a 1.71b 1.55b 1.31b Trichoderma 2.86a 2.66ab 2.41b 1.84b 1.61b 1.48b P CV (%) The chemical fungicide application was not significantly different in disease index when compare to GAP, Ketomium and Trichoderma biofungicide.

40 Results Figure Citrus tree in the non-treated control for 9 months Before application After application T 1- control non treated

41 Results Figure Citrus tree in chemical fungicide treatment (metalaxyl) for 9 months Before application After application T 2- Chemical fungicide (metalaxyl)

42 Results Figure Citrus tree in good agricultural practice treatment (Ketomium-biofungicide + metalaxyl) for 9 months Before application After application T 3 chemical fungicide and biofungicide (GAP)

43 Results Figure Citrus tree in Ketomium-biofungicide treatment for 9 months Before application After application T 4 = Chaetomium (biofungicide)

44 Results Figure Citrus tree in Trichoderma-biofungicide treatment for 9 months Before application After application T 5 = Trichoderma (biofungicide)

45 Conclusions The Pythium ultimum isolates were proved to be pathogenic to cause citrus root rot by detached leave method and inoculated seedling test. C. cupreum BTB 06, C. globosum BTB 014 and Trichoderma harzianium BTB 022 were selected to be suitable fungi against Pythium ultimum because they could inhibit the mycelia growth of P. ultimum over 50% in dual culture.

46 Conclusions Three effective antagonistic fungi could inhibit the colony of Pythium ultimum T41 with percent growth of inhibition (PGI) as follows: Trichoderma harzianium BTB 022 (60.33%), Chaetomium globosum BTB 014 (66.44%) and Chaetomium cupreum BTB 06 (70.88%). The chemical fungicide-metalaxyl was not significantly differences in disease reduction when compared to GAP, Ketomium and Trichoderma biological fungicides.

47 Conclusions The biological fungicides expressed a curative effect for disease control and it would be good if the application of biological fungicides be applied for protecting in the healthy plants. It was clearly indicated that the tested biological products of Chaetomium, Trichoderma and chemical control (metalaxyl) were significantly different to control Pythium root rot when compared to the non-treated one (control).

48 Recommendations The three antagonist fungi should be tested in a wide area in Cambodia The citrus plantation need to apply bio- fungicides to prevent the root rot diseases and should be treated in earlier stage.

49