Identification of culturable endophytes isolated from apple tissues with antagonism towards Neonectria ditissima Jing Liu, Hayley Ridgway & Eirian Jones
Background Apple production in NZ widely cultivated $720M export crop in 2016 environmentally friendly cultivation IFP since the 1990 s Royal Gala Braeburn Fuji Smitten Mariri-red Red-delicious European canker (Neonectria ditissima) leading cause of apple trunk disease attack buds, shoots, spurs and branches loss of trees (canker on stem) yield loss (blossom-end and stem-end fruit rots)
European canker (N. ditissima) a blistering on the bark Symptoms concentric rings of cracking Wound parasite Spores flaky bark vascular staining below the canker Leaf scar Sporodochia (conidia) Fruit scar leaf and fruit scars wounds caused by pruning, insects/pathogens, frost injury Perithecia (ascospores)
Control Strategies Current strategies Time and labour consuming No curative fungicides Fungicide resistance/residue risk Field inspection Pruning Protectant fungicides Biological control Sustainable Reduced fungicide resistance/residue risk Limited non-target impacts Endophytes Occupy the same niche as the pathogen Provide long term control by colonizing & protecting wounds Culturable endophytes from apple as biocontrol agents against N. ditissima
Sampling Endophytes isolated from healthy apple shoots 2 nd leaves green stem (~7 cm) woody stem (~7 cm) 3 rd leaves Main samplings Region Variety Spring 2015 Nelson (South Island) Royal Gala, Braeburn, Jazz and Envy. Hawke s Bay (North Island) 35 heritage varieties (Plant & Food Research, Hawke s Bay) Autumn 2016 Nelson Royal Gala, Braeburn
Research methods Endophyte isolation In vitro dual culture screening ID by PCR sequencing Functional assays Surface sterilisation Isolation Culture collection Inhibition effect 16S rrna (bacteria) ITS (fungi) Filtrate assay Volatile assay Siderophore production assay
Results Antagonistic endophytes found 1004 bacteria - 18 inhibited N. ditissima by >20% 81 fungi (33 morphotypes) - 18 (10 morphotypes) inhibited N. ditissima growth 1 actinobacterium recovered Identification Bacteria = Bacillus spp. (n=9) Pseudomonas spp. (n=9) Fungi = Epicoccum spp. (n=5) Biscogniauxia spp. (n=4) Chaetomium spp. (n=3) Phylctema spp. (n=2) Diaporthe sp. (n=1) Neoseptophoma sp. (n=1) Penicillium sp. (n=1) Unidentified sp. (n=1) *Genera of known pathogens or unidentified species discarded from future work
Inhibition of N. distissima by antagonistic bacteria Phase 1 - Primary dual culture screening 3 test bacteria per plate 1 positive control (+) Pathogen inoculated in centre Inhibitory isolates selected for second phase Test Plate + Control Phase 2 - Percent inhibition 1 bacterium per plate 4 pseudo replicates per plate Inhibition % calculated 77% 44% Control
Inhibition effect of the antagonistic fungi Inhibition types endophyte (E) either co-inoculated on same day or inoculated 10 days before/after inoculating the pathogen (P) Overgrowth (6 isolates) Inhibition zone (10 isolates) P E P E P E same day 10 days later Unclear inhibition zone (2 isolates) same day P E P E P E P E same day 10 days later same day 10 days prior
Siderophore production by endophytes Inhibits pathogen by chelating iron (essential element) Chrome Azurol S (CAS) agar did not support Bacillus spp. (Gram + ) growth Modified CAS (MCAS) assay Bacterium Endophyte Siderophore production CAS reaction (mm/day) Pseudomonas spp. (7) + 0.9-3.7 Bacillus spp. (8) + 0.1-1.1 Bacillus spp. (1) - 0.0 Nutrient agar CAS reaction CAS agar Epicoccum spp.(5) +(5) 0.4-1.3 Biscogniauxia spp. (4) +(3), -(1) 0.0-0.8 Chaetomium spp. (3) +(2), -(1) 0.0-0.8 Neoseptophoma sp. (1) + 0.5 Penicillium sp. (1) + 1.4 Fungus PDA CAS reaction CAS agar
Conclusions Percentage of antagonistic bacteria was low = 1.8% Bacteria (n=18) 89% (n=16) from commercial cultivars out of 870 isolates Royal Gala [9], Braeburn [5] and Jazz [2] 11% (n=2) from a heritage variety (Grimes Golden) out of 134 isolates 28% (n=5) from commercial varieties in autumn sampling out of 58 isolates Royal Gala [4], Braeburn [1] Fungi (n=18) 67% (n=12) isolated from commercial varieties sampled in 14 IFP managed orchards 33% (n=6) isolated from commercial varieties in 2 organic orchards Antagonistic bacteria were more frequent in the autumn sampling. A higher percentage of antagonistic fungi were isolated from organic orchards.
All bacteria and 83% fungi were from low EC infection blocks (infection rate 30%) May indicate beneficial endophytes contribute to low EC infection. All antagonistic bacteria belonged to Bacillus spp. and Pseudomonas spp. The antagonistic fungi contained two known biocontrol genera Epicoccum and Chaetomium Pseudomonas spp. produced more siderophores than Bacillus spp. Penicillium sp. produced the most siderophores amongst the 14 test fungi In summary, endophytes isolated from apple tissues have the potential for use in sustainable control of N. ditissima.
Future work Endophyte colonisation in detached and attached apple shoots Four out of six tested bacterial isolates endophytically colonised detached apple stems In vivo biocontrol activity assays of the selected endophytic isolates Source of pictures NZ apple varieties: yummyfruit.co.nz European canker (N. ditissima) symptoms: http://www.pipfruitnz.co.nz Sporodochia, perithecia structures: http://www.pipfruitnz.co.nz Current control strategies: http://www.pipfruitnz.co.nz
Acknowledgements Pipfruit NZ and Lincoln University for funding Tim Herman (Pipfruit NZ Industry liaison) for advice Orchard owners and Plant &Food Research for access to sample ICPP-2003 travel fund for funding conference travel SPPH 2017 conference organisers
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