Biological control of walnut blight: screening of antagonistic bacteria for Xanthomonas arboricola pv. juglandis and evaluation of their efficacy

Transcription

1 Biological control of walnut blight: screening of antagonistic bacteria for Xanthomonas arboricola pv. juglandis and evaluation of their efficacy Hatice OZAKTAN Mine YAVAS Ahmet AKKÖPRÜ Emek ASLAN Adem BOZKURT Department of Plant protection, Faculty of Agriculture, University of Ege, Bornova, İzmir / TURKEY hatice.ozaktan@ege.edu.tr

2 Walnut plantations can be observed in each region of Turkey. In deed, Commercial walnut orchards has been started to establish in Turkey in 1990s.

3 The most common walnut cultivars growing in Turkey are local varieties such as Yalova, Şebin, Bilecik, KR and Kaman.

4 The production of walnut transplants of Turkey is approximately million transplants per year. An appearence from walnut nurseries of Sahil Yenice Village in Marmara Region

5 BACTERIAL BLIGHT OF WALNUT IN TURKEY Since walnut has been cultivated in Turkey bacterial blight has occured. We have isolated and identified some strains of Xanthomonas arboricola pv.juglandis in most of walnut growing areas of Turkey since 2007 year. Bacterial blight of walnut was very destructive in Turkey since frost injury and rainy weather conditions and then warm temperatures coincided with the budbreak period in Marmara Region of Turkey was observed during last spring period. Xanthomonas arboricola pv.juglandis caused damages all of the tree soft organs in spring of 2007: male and female flowers, nutlets, leaves and fruitfull buds bearing shoots.

6 Infected catkins and buds turned black

7 Shoot blight symptoms

8 Infected flowers and young tissues were died and shrivelled

9 IMMATURE NUT INFECTION Severe immature fruit drops have been observed on some walnut cultivars because of bacterial blight infection

10 Because of the bud infection of Xaj during early blooming period, walnut blight causes serious reduction in fruit yield and tree vigor

11 Aim of this research: Copper based compounds have been the only means of control of Walnut bacterial blight induced by Xanthomonas arboricola pv.juglandis for more than 40 years. Data indicates that copper resistant strains of the walnut blight pathogen are not killed by standard copper applications under field conditions. Biological control of walnut blight pathogen may be alternative to chemical control. The goal of this study was to determine whether bacterial antagonists could be used to control of X.a.pv.juglandis ( the causal agent of bacterial blight on walnut).

12 MATERIAL AND METHODS OF THIS STUDY

13 Tested Bacterial Pathogen: The most virulent strain of Xanthomonas arboricola pv.juglandis (W7/1), isolated from diseased buds of walnut trees in Izmir, was used for the inoculation. The bacterium was grown on King s B medium at 24 o C, for 48h. Bacteria were scraped and suspended in distiled water. The final concentration was spectrophotometically adjusted to 5x10 8 cfu/ml on the basis of the absorbancy at 600 nm.

14 Tested Bacterial Antagonists: Totally, 35 bacterial antagonists were screened for their in vitro biocontrol activity to X.arboricola pv.juglandis. A collection of 29 bacterial strains were isolated as epiphytes from phylloplane of healthy walnut trees. The others had been previously isolated from beans, cucurbits, and olive trees and found promising when they tested in different host X pathogen combinations in Turkey. Pantoae agglomerans strain C9/1 and Pseudomonas fluorescens strain A506 were also tested as reference biocontrol agents against X.a.pv.juglandis. Strains were characterized on basis of Gram response, fluorescence on King B agar and some physiological tests. Approximately 52% were identified as fluorescent pseudomonads, 28% Gram Positive bacteria, and 20% Gram Negative bacteria 28% 20% FP G(+) G(-) Tested bacterial antagonists for in vitro antagonistic activity against X.a.pv.juglandis 52%

15 In vitro Antagonism assay: Tests for antibiosis between antagonistic bacteria and X.arboricola pv.juglandis were conducted in TSA plates. 100 µl of 10 8 CFU/ml suspension of X.arboricola pv.juglandis strain W7/1 was spread over the agar surface and dried.

16 In vitro Antagonism assay: Four sterile assay disks (5 mm diameter) were placed on the agar and a suspension of each candidate antagonistic bacteria (10 9 CFU/ml, 10 µl /disk) was dropped onto each disk. Plates were incubated at 24 0 C for 48h to 72h and examined for signs of clear zones indicating growth inhibition and recorded as mm. There were four replications (or agar plate) per each antagonistic bacterium. The tests were done twice.

17 Immature nut test for biocontrol activity of bacterial antagonists Immature nuts were collected days after nut set (Gf) Symptomless, detached walnut fruits (susceptible local cultivar Kaman 1 to Xaj) were surface disinfected with SDW before inoculation Immature nuts were submitted to inoculation of antagonistic bacteria and then pathogenic bacteria (Xaj) on marked areas of nuts under controlled environmental conditions

18 Immature nut test for biocontrol activity of bacterial antagonists The antagonistic bacteia were grown on King s B medium at 24 o C, for 24h. Bacteria were scraped and suspended in distilled water. The final concentration was spectrophotometically adjusted to 5x10 9 cfu/ml on the basis of the absorbancy at 600 nm The antagonistic bacteria were infiltrated into marked areas on the surfece of fruits as 30µl per each area Suspension of antagonistic bacteria

19 Immature nut test for biocontrol activity of bacterial antagonists One hour later, pathogenic bacteria noculation was carried out by infiltration of a suspension 10 8 cfu/ml of a previously selected virulent strain of X.a.pv.juglandis into same marked areas on the nuts. Four inoculations of 30µl of bacterial suspension were performed for each nut and eight replicates of 2 nuts per cultivar were tested. Sterile Distilled water (SDW) was injected into the immature nuts as Control Negative treatment Pathogenic bacteria were infiltrated into marked areas on the nuts after SDW application as Control Positive treatment Suspension of pathogenic bacteria (Xaj)

20 Immature nut test for biocontrol activity of bacterial antagonists Pantoae agglomerans strain C9/1 was also tested as reference biocontrol agent against X.a.j. The incubation lasted 5 days at 24 0 C, RH over 90%. Bacterial blight incidence on immature walnut fruits were evaluated by 0-4 scale

21 0-4 SCALE : No symptoms 1: very small superficial points on the inoculation point 2: Less then 2 mm, supreficial and small spots on the inoculation point 3: Blackening on the inoculation point of nut by 2 mm to 5 mm, and extending the inner tissues by 1mm 4: Blackening on the inoculation point of nut more than 5 mm, and extending the inner tissues by 2mm to 4 mm

22 Walnut seedling test for biocontrol activity of bacterial antagonists to Xaj Bacterial antagonist strains, which were effectively inhibited the development of Xaj on immature nuts were selected for walnut seedling pot test One year-old walnut seedlings of cv.chandler, which is susceptible to Xaj, were used for biocontrol activity test of bacterial antagonists All grafting wood was whip grafted onto one year-old Juglans regia seedlings, and grafted seedlings were potted and placed in a greenhouse before inoculation of bacteria.

23 Walnut seedling test for biocontrol activity of bacterial antagonists to Xaj In the mid of August 2008, the potted walnut trees were applied with selected antagonistic bacterial suspensions (10 9 cfu/ml) by spraying the leaves, Walnut seedlings were treated with the reference chemical copper-oxychloride + maneb (0.2%,37,5%+20% a.i.) to compare with the efficacy of bacterial antagonists Pantoae agglomerans C9/1 was also applied as referance biocontrol strain. Walnut trees were inoculated with the suspension of Xaj strain W7/1 (10 8 cfu/ml) 24h after treatments Water treated trees were included as Negative Control. Each treatment was applied to four walnut seedlings in randomized complete blocks.

24 Walnut seedling test for biocontrol activity of bacterial antagonists to Xaj Pots were covered with plastic bags and kept in an air-conditioned room. Temperature and humidity conditions were 24 0 C and 90-95%, respectively. Walnut seedlings were evaluated for Xaj damages in leaves, depending on the symptom development on the leaves after pathogen challenge.

25 Disease symptoms on leaves were assessed according to a 0-5 scale 1 = yellowish halos without necrosis 2 = necrotic specks 0 = healthy 3 = three necrotic spots or fewer 4 = more than three necrotic spots 5 = with large areas of necrosis

26 RESULTS In vitro antagonism test results In vivo test results Immature nut test results for biocontrol activity of bacterial antagonists Walnut seedling test for biocontrol activity of bacterial antagonists to Xaj

27 In vitro antagonism test results Totally, 35 bacterial antagonists were screened for their in vitro biocontrol activity to X.arboricola pv.juglandis. Out of ten of 29 bacterial antagonist strains isolated from healthy walnut trees inhibited Xaj with zones of inhibition ranged from 4 mm to 13 mm in vitro tests. According to in vitro test results, the most efficient strain was fluorescent Pseudomonad strain WH45/1b, producing by 13 mm inhibition zone against Xaj, which was isolated from healthy walnut leaves.

28 In vitro antagonism test results The Gram (+) antagonistic strain WH85/2, isolated from healthy buds of walnut trees was second promising antagonistic bacterium producing 10 mm inhibition zone against Xaj on agar plate.

29 In vitro antagonism test results Nineteen bacterial strains of tested 29 bacterial antagonist candidates from healthy walnut trees couldn t produce any inhibition zones against Xaj in in vitro test. Reference biocontrol strains, P.fluorescens strain A506 and P.agglomerans C9/1 couldn t show any antibiosis against Xaj on TSA plate. The other antagonistic bacteria, had been previously isolated from different plants and found promising when they tested in different host X pathogen combinations in Turkey couldn t also produce any inhibition zones against Xaj on TSA.

30 WH 77/1 WH 84/2 WH 85/1 WH85/2 WH 93/2 WH 95/2 WH 97/1 WH 31/1 WH 43/1b WH 45/1b WH 56/1 WH 67 WH 68 WH39/1a WH 48/1a WH 54/1 WH 42/1a WH 55/4 C 9/1 K (+) K (-) Disease severity of infected nuts % a a a ab abc abc abc bcd bcd bcd cde cde def cdef def ef ef ef ef f biocontrol activity of tested bacterial antagonists against Xaj on immature nut test Of the 35 FP strains tested, 18 inhibiting the growth of Xaj between 3.00 mm to mm on TSA plate were selected and tested for their ability to control of bacterial blight of walnut on the immature nut test. Approximately 39% of the antagonistic bacterial strains tested on immature nut test, significantly reduced bacterial blight of walnut by 73% to 88% compared to the pathogen-alone treatment tested bacterial antagonists

31 biocontrol activity of tested bacterial antagonists against Xaj on immature nut test Bacterial antagonist strains WH48/1A, WH39/1A, and WH68 were the most effective for inhibition of Xaj on immature nuts giving 88%, 84% and 86% reduction, respectively

32 biocontrol activity of tested bacterial antagonists against Xaj on immature nut test Bacterial antagonist strains WH77/1, WH85/2, and WH67 successively inhibited the growth of Xaj on immature nuts by the rate of 73%, compared to the pathogen alone treatment.

33 biocontrol activity of tested bacterial antagonists against Xaj on immature nut test Bacterial antagonist strains WH43/1B and WH31/1 inhibited the bacterial blight incidence on detached walnut fruits less then 20%, compared to the pathogen alone tratment and found less effective strains agaist Xaj

34 biocontrol activity of tested bacterial antagonists against Xaj on immature nut test These bacterial strains were completely ineffective to inhibit the growth of Xaj on imature nuts

35 Walnut seedling test results

36 Biocontrol activity of tested bacterial antagonists against Xaj on walnut seedling test % WH 77/1 WH 43/1b WH 55/4 WH 48/1 a WH 39/1a WH 68 WH 85/2 WH 45/1 b WH 67 P. aglomerans C9/1 Control Pozitive Cuppered chemical Control Negative Mean Disease Severity on Walnut leaves (%) Efficacy (%) Bacterial strains WH77/1, WH43/1B, WH55/4 and WH48/1A were the most effective for inhibition of Xaj on seedling test giving 89%, 78%, 79% and 76% disease reduction on walnut leaves, respectively.

37 Walnut seedling test for biocontrol activity of bacterial antagonists to Xaj KONTROL (+) EFFECTIVE BACTERIAL WH 48/1A ANTAGONISTS WH 77/1 TO XAJ

38 WH55/4 CONTROL + EFFECTIVE BACTERIAL ANTAGONISTS TO XAJ ON SEEDLING TEST

39 CONTROL (+) P.agglomerans strain C9/1, which was tested for Xaj as reference biocontrol agent, and Cuppered chemical reduced the bacterial blight of walnut leaves on seedlings by 51% and 45%, respectively.

40 BACTERIAL STRAINS WH45/1B AND WH67 INHIBITED THE SYMPTOM DEVELOPMENT ON LEAVES OF WALNUT SEEDLING BY THE RATE OF 45% AND 16%, RESPECTIVELY

41 In vitro and in vivo biocontrol effects of some antagonistic bacterial strains against X.a.pv.juglandis, causal agent of walnut blight Treatments In vitro biocontrol activity (inhibition zones as mm) Efficacy (%) (Detached walnut test results ) Efficacy (%) (Walnut seedling Trial results ) WH 77/1 6, WH 43/1B 7,7-78 WH 55/ WH 48/1A 2, WH 39/1A WH 68 6, WH 85/2 9, WH 45/1B 12, WH 67 4, P.agglomerans C9/ Cupperted Chemical Control Positive Control Negative

42 In conclusion: Because of the development of copper-resistant pathogen populations, management of walnut blight is dependent on applications with higher rates of copper, addition of other bactericidal compounds to copper treatments (e.g., Kocide -Manex ) Potential alternatives including bacterial antagonists and other bactericidal products (Serenade), and systemic acquired host resistance (SAR) compounds may be proposed as control measures for bacterial blight of walnut. According to the immature nut assay and seedling test, some strains of Fluorescent Pseudomonads and Gram (+) bacteria were found promising in respect to biocontrol of bacterial blight of walnut caused by Xaj P.agglomerans strain C9/1, which was tested for Xaj as reference biocontrol agent, reduced the bacterial blight of walnut on immature nuts by 40% and on leaves of walnut seedlings by 51%, whereas it couldn t produce any inhibition zones against Xaj

43 In conclusion: It has been suggested that antagonistic bacteria isolated from the healthy walnut orchards might be better adapted to that crop and provide better control of bacterial blight of walnut than organisms originally isolated from other plant species. To screen the promising biocontrol agents against Xaj, detached immature nut assay is a quick and reliable method According to the in vivo biocontrol test results on walnut seedlings (cv.chandler), most of the tested bacterial antagonists significantly reduced the symptom development on the walnut leaves by 49% to 89% compared to the pathogen-alone treatment. The results obtained from both nut test and seddling test were found correlative, except for the strain WH 43/1B. Further studies are needed to know the exact mechanism of action of bacterial antagonists in controlling the bacterial blight of walnut.

44 THANK YOU FOR YOUR ATTENTION