Potential new tools for combating brown rot: the milk protein story Kirstin Wurms, Peter Wood, Philip Elmer, Annette Ah Chee, Megan Callaghan, Paul Harris, Frank Parry, and Tom Wheeler
Why look for new tools for brown rot control? Under the right conditions brown rot is the most difficult summerfruit disease to control. Heavy reliance on synthetic pesticides (SPs) SP use becoming more restricted: - Part of a global shift to reduce residue risks - Human + environmental health concerns - Ongoing issues with fungicide resistance - Removal of products from spray recommendations Highlights the need for safer, natural-product based alternatives to SPs
Advantages of natural products (NPs) Ease of product registration e.g. dairy products considered non-toxic to humans Safe for the environment Short withholding periods Suitable for conventional growers and the expanding organic market (extended market size) Often have multiple modes of action May offer eradicant activity
Disadvantages of NPs Handling problems/formulation issues Phytotoxicity Spoilage/smell Blockage of spray equipment Poor durability &/or inconsistent control Lack of a unique selling point Growth of non-target organisms e.g. sooty mould
Why chose minor milk proteins (MMPs)? Sprays of milk/whey have controlled powdery mildew in the lab & field (e.g. Bettiol 1999 and Crisp et al. 2006). These products are nutritionally complex encourages growth of non target organisms (e.g. sooty mould) variable results. Activity of MMPs against plant pathogens is unknown. Our prelim. lab assays showed potential of MMPs as novel sources of natural-product biopesticides. Bettiol W. (1999). Effectiveness of cows milk against zucchini squash powdery mildew in greenhouses. Crop Protection 18: 489-492. Crisp P., Wicks T.J., Lorimer M., Scott E.S. (2006). An evaluation of biological and abiotic controls for grapevine powdery mildew. Parts 1 &2. Australian Journal of Grape & Wine Research 12: 192-211.
Direct growth inhibition studies (1) Initially five different MMPs were tested in multi-well plate assays against four globally important plant pathogens. Three MMPs (MMP1,2,3) were selected for further studies against Monilinia fructicola (MF). Growth inhibition assays in multi-well plates have looked at: - Efficacy of different concentrations of MMPs - Growth inhibition versus biocidal activity
Direct growth inhibition studies (2) OD 0.45 0.40 0.35 0.30 0.25 MMP1vs. Monilinia fructicola 100ug/ml 50 25 12.5 6.3 CONT 0.20 0.15 0.10 0 16 24 40 48 64 72 88 96 hrs Incubation time
Direct growth inhibition studies (3) OD 0.40 0.35 0.30 0.25 0.20 MMP1 vs Botrytis cinerea 2000ug/ml 1000 500 250 125 63 Cont 0.15 0.10 0 17 24 41 48 64 72 88 96 hr Incubation time
Direct growth inhibition studies (4) MMP 1,2,3 were also effective against Septoria sp. (leaf spot pathogens) MMPs 1,2,3 were active against Elsinoe pyri, and MMP1 & 3 against Venturia ineaqualis (apple pathogens). All three MMPs also had moderate to high antimicrobial activity against a range of food and wine spoilage micro-organisms: Septoria Elsinoe Venturia
Efficacy studies on plants Multi-well plate assays were not carried out on plant tissue. The next step was to demonstrate activity on plant tissue in smallerscale lab experiments before progressing to the field. Two types of plant tissue were tested in lab trials peach blossoms and cherry fruits.
Peach blossom assay: methods (1) Unopened Golden Queen peach blossom shoots were collected from an unsprayed block at Havelock North Research centre. These were graded, sanitised, placed in a culture solution, forced into flower, sprayed with MMP solutions (1 mg/ml) then dried, before misting with a spore suspension (2500 conidia/ml) of M. fructicola (MF).
Peach blossom assay: methods (2) The shoots were incubated for 6 days at 22⁰C in high humidity chambers Simulates high disease pressure conditions Assessed for % blossom blight and % flowers with sporulation
Peach blossom assay: results (1) Percent blossom blight after minor milk protein treatment (MMP 1, 2 3) & MF inoculation MF only Fungicide MMP1 MMP2 MMP3 80 % 0 % 42 % 47 % 49 % Least Significant Difference = 22% MF = a liquid spore suspension of 2,000 conidia/ml Fungicide = Rovral Flo
Peach blossom assay: results (2)
Peach blossom assay A very tough test for any natural product Severe brown rot infection period for six continuous days Results on detached peach shoots were encouraging Significant anti-fungal activity demonstrated No obvious phytotoxicity observed Need for studies of brown rot control on fruit
Cherry fruit assay: methods Sweet Valentine cherries from Clyde Research Centre were dipped in MMPs (1 mg/ml), dried, then sprayed with a liquid suspension of M. fructicola spores (100,000 conidia/ml). Once dry, cherries were spaced out individually on trays, & incubated for 6 days at 22-23⁰C in humid chambers. Severity of brown rot infection = proportion of each individual fruit surface covered in spores.
fect Cherry fruit assay: results (1)
Cherry fruit assay: results (2) MF only Rovral MMP1
Golden Queen field trial: methods (1) Mature Golden Queen trees from the research centre orchard in Havelock North. Five trees/treatment Fungicides early season Three MMP spray applications at 7 day intervals (21,14 & 7 d pre-harvest) of low, medium and high doses of MMP1, MMP3 and MMP X.
Golden Queen field trial: methods (2) Disease assessment made just before harvest = % rotten fruit. At harvest fruit were placed in cold storage for 3 weeks. % rots assessed after removal from cold storage, and after 3 d at 20ºC to induce any rot symptoms.
Golden Queen field trial: pre-harvest results X
Golden Queen field trial: postharvest assessments X X
Overall conclusions MMP1, MMP3, and MMP X are the most effective minor milk proteins against brown rot. Efficacy demonstrated in the lab & field, on flowers (peaches) & fruit (peaches & cherries). Prelim. lab results suggest antimicrobial activity against a range of other pathogens, including Botrytis cinerea.
Future research Expand trials on Botrytis cinerea for cherries (incl. applications over flowering & pre-harvest to assist with fungicide resistance mgt) Research to verify that allergens are not present Testing of UV instability and/or UV enhancement Further testing of spectrum of activity Take home message: MMP are environmentally friendly, natural proteins that have shown effective brown rot control against cherries and peaches in the lab and the field. Further research is needed if they are become a commercial reality.
Acknowledgements AgResearch and Plant & Food Research and the Agricultural Marketing and Research and Development Trust (AGMARDT) for funding this research Tatua for kindly supplying milk proteins Maureen George and Jennifer Parry for ably assisting with plant tissue assays Claire Hall for excellent business management of the programme. Summerfruit NZ for allowing me to present this work at their annual conference
Kirstin.Wurms@plantandfood.co.nz www.plantandfood.co.nz