Clarke et al. Dry heat for grape phylloxera disinfestation 1 Dry heat as a disinfestation treatment against genetically diverse strains of grape phylloxera C.W. CLARKE1 , S. NORNG2, D. YUANPENG3, B.M. CARMODY1 and K.S. POWELL1* 1 Agriculture Victoria, Rutherglen, Vic., 3685, Australia; 2 Department of Economic Development, Jobs, Transport and Resources, Melbourne, Vic., 3053, Australia; 3 College of Horticulture Science and Engineering, Shandong Agricultural University, Key Laboratory of Crop Biology of Shandong Province, Tai’an, Shandong, 271018, China *Present address: Sugar Research Australia, Meringa, Qld 4865, Australia. Corresponding authors: Dr Catherine Clarke, email catherine.clarke@ecodev.vic.gov.au and Dr Kevin Powell, email kpowell@sugarresearch.com.au Abstract Background and Aims: Grape phylloxera, Daktulosphaira vitifoliae (Fitch), causes damage to ungrafted European grapevine, Vitis vinifera L. worldwide. In Australia, 83 phylloxera genetic strains exist in distinct zones and the primary management strategy is quarantine. While moving viticultural machinery, particularly harvesters from a phylloxera infested zone, dry heat treatment at 40 and 45 C for 75 and 120 min, respectively, is recommended. Methods and Results: First instars of six root-feeding phylloxera, G1, G4, G7, G19, G20 and G30, were subjected to dry heat treatment at 22, 35, 40 and 45 C for 75, 90 and 120 min. For G20 and G30, first instars were subjected to 40 C for 135 min in a separate treatment. Across the six phylloxera genetic strains, no phylloxera survived treatment at 45 C for 75 min. First instars of G1, G4, G7, G19 phylloxera did not survive treatment at 40 C for 120 min. For G20 and G30 phylloxera, however, 100% mortality at 40 C was achieved only when time of treatment was increased to 135 min. The development of surviving phylloxera on excised V. vinifera cv. Chardonnay roots was influenced by the temperature of the dry heat treatment. Conclusions: Results validated the effectiveness of dry heat disinfestation protocol of 45 C for 75 min across diverse phylloxera genetic strains. The alternative protocol of 40 C for 120 min was not effective across all phylloxera strains and a duration of 135 min is recommended. Significance of the Study: This study highlights the relative sensitivity of genetically diverse phylloxera to heat treatment and duration of exposure. Keywords: dry heat, genetic strain, grape phylloxera, quarantine, viticultural machinery Introduction In Australia grape phylloxera, Daktulosphaira vitifoliae (Fitch) is listed among the top 10 economically important grapevine pests (Schofield and Morison 2010). Although widespread in most grapegrowing regions, phylloxera is currently confined to phylloxera infested zones (PIZs) in parts of Victoria and New South Wales (Powell 2008). The pest is not known to be present in other Australian states and territories and these areas are therefore considered as either phylloxera exclusion zones (PEZs) or phylloxera risk zones (PRZs) (Vine Health Australia 2017). Grape phylloxera is, therefore, an important endemic biosecurity pest for the Australian viticulture industry. Effective disinfestation protocols that limit the risk of phylloxera transfer between quarantine zones, particularly on viticulture machinery, are required. In Australia, grape phylloxera lives and feeds almost exclusively on the roots of grapevines and some genetic strains can occasionally be found in distinctive galls on leaves (Kellow et al. 2004). First instars are the most active stage and are present above and below the soil surface. [Correction added on 2 March 2018, after first online publication: Affiliation 1 has been corrected to ‘Agriculture Victoria, Rutherglen, Vic., 3685, Australia.’] doi: 10.1111/ajgw.12340 © 2018 Australian Society of Viticulture and Oenology Inc. Below ground, first instars locate suitable host material and establish feeding sites on roots, develop into intermediate instars and adults while sucking sap from grapevine parenchymal cells. Root-feeding damage causes the development of yellow fleshy galls (nodosities) on immature non-lignified roots and root swellings (tuberosities) on mature lignified roots (Kellow et al. 2004). Damage to the roots also leads to secondary root necrosis because entry of fungal pathogens leading to decay and loss of functioning non-lignified roots and reduced grapevine vigour (Omer and Granett 2000). As a result of root damage, symptoms of phylloxera infestation are expressed above ground as low canopy vigour, premature leaf yellowing and reduced bunch size (Powell et al. 2013). Phylloxera first instars are active above ground in spring and summer and have been detected on grapes, foliage (Powell et al. 2000, Deretic et al. 2003) and grape harvesters (King and Buchanan 1986). Consequently, dry heat treatment is a recommended disinfestation treatment against phylloxera on vineyard machinery (National Vine Health Steering Committee 2009). Before leaving a PIZ or a PRZ to a PEZ, vineyard machinery is subjected to dry heat at 40 or 45 C for 120 or 75 min, respectively. The effectiveness of dry heat treatment has previously been verified against G1 2 Dry heat for grape phylloxera disinfestation and G4, two genetically similar phylloxera strains (Korosi et al. 2012). Recent studies by Clarke et al. (2017a, b), however, revealed variations in survival of genetically diverse phylloxera when subjected to both chemical and hot water treatments. Thus, this study compared the effectiveness of dry heat disinfestation against genetically diverse phylloxera strains. Materials and methods Maintenance of phylloxera strains Six phylloxera strains, G1, G4, G7, G19, G20 and G30, initially collected from ungrafted Vitis vinifera in commercial vineyards in central and north-east Victoria, Australia, were used in the experiments. The phylloxera strain G1, was collected from central Victoria in the Maroondah PIZ and all other genetic strains were sourced from north-east PIZ in Victoria. The strain G4 was collected from the King Valley, G7, G19 and G30 from Rutherglen and G20 from the Buckland Valley. All phylloxera strains were genetically characterised using six mitochondrial markers as described in Umina et al. (2007). The insects were mass reared on excised V. vinifera cv. Chardonnay roots as described in Kingston et al. (2007). Eggs of each phylloxera strain were collected from the excised root stock cultures with a small artists’ paintbrush and separately placed on moistened filter paper in a 90 × 25 mm Petri dish. The Petri dish was sealed with cling film (Rapfast PVC food packaging, Integrated Packaging, Reservoir, Vic., Australia) to create an egg hatching chamber (Clarke et al. 2017a). The eggs were incubated at 22 C until first instars hatched. Active 1-day-old first instars from each of the genetic strains were collected and used in the experiments. Dry heat treatments Ten 1-day-old first instar phylloxera of either G1, G4, G7, G19, G20 or G30 were placed in a treatment vial measuring 5.5 cm high and 2.5 cm in diameter (Korosi et al. 2012). The vial was then placed in a sealed cylindrical plastic container that was partially filled with saturated magnesium chloride solution to create an environmental chamber with 30% relative humidity (Buchanan 1990, Korosi et al. 2012). The RH of 30% was chosen as this is the typical level reached in a commercial dry heat shed at ≥40 C (Dr Kevin Powell, pers. comm., 2017). The environmental chamber with the vial containing phylloxera was randomly assigned to one of three shelves of a fan-forced oven (Qualtex Model Number OM24SE3D, FSE Scientific, Marrickville, NSW, Australia) set at 22, 35, 40 or 45 C for either 75, 90 or 120 min. A proportion of first instars of G20 and G30 phylloxera survived in 40 C for 120 min, thus a treatment of 135 min at 40 C was conducted separately. The treatments were replicated five times for each combination of phylloxera genetic strain, temperature and time. The experiments employed a randomised block design where a replicate block for each temperature × time treatment was all initiated at the same time with first instars that emerged from the same batch of eggs. Only eggs that were produced within 24 h were used for the experiments. The oven temperature on each of the three shelves (subblock) was monitored every 30 s using a Gemini data logger (Tiny Tag Explorer, Hastings Data Loggers, Port Macquarie, NSW, Australia) and temperature fluctuations averaged 0.8 C for each temperature and treatment duration. Following treatment, first instars were removed from the vials with a fine artists’ paintbrush, placed onto a filter Australian Journal of Grape and Wine Research 2018 paper and examined under a low power magnification microscope. First instars were categorised as alive if there was leg and/or antennal movement and dead if there was no movement when gently stimulated with the tip of a paint brush up to 2 h after treatment. A proportion of first instars that survived treatments at 22, 35 and 40 C were placed on excised V. vinifera Chardonnay roots to examine posttreatment effects on establishment, development and subsequent reproduction. For each genetic strain, first instars that survived the individual heat treatments at 22, 35 and 40 C for 75, 90, 120 min, were pooled onto a single root piece in a 90 × 25 mm Petri dish. Phylloxera survival on the root pieces was checked daily for the first 3 days and weekly thereafter. First instars that established feeding sites on the roots, that died prematurely and that developed to adulthood and reproduced were counted and recorded. Statistical analysis The proportion of live insects was analysed using an ANOVA in GenStat (GenStat Release 18) (VSN International, Hemel Hempstead, England). The treatment structure was specified as phylloxera genetic strain × time × temperature. The blocking structure was specified as vial nested within environmental chamber nested within the replicate (blocks). This was written in GenStat as Replicate/Chamber/ Vial. The proportion of first instars that established feeding sites, died prematurely or developed to adults and subsequently laid eggs was analysed using ANOVA (unbalanced design using regression). Fishers protected least significant difference (LSD; P = 0.05) was used to separate means where significant. Results and discussion The upper thermal limit for 100% mortality was achieved at 45 C when first instars across all six phylloxera genetic strains were subjected to the heat treatment for a duration of 75 min (Figure 1). The results thus validated the existing disinfestation protocol of 45 C for 75 min (National Vine Health Steering Committee 2009). For the alternative recommended protocol of 40 C for 2 h (National Vine Health Steering Committee 2009), 100% mortality was reached after 120 min exposure for G1, G4, G7 and G19 phylloxera (Figure 1), however, and 2 and 3% first instars of G20 and G30, respectively, required 135 min to achieve 0% survival (Figure 1). Overall survival was reduced as temperature and time of exposure increased, a trend that was consistent across the six phylloxera genetic strains (P < 0.001; Figure 1). When subjected to dry heat at 40 C for 75 min, first instars of G19 phylloxera were relatively resistant to the treatment compared to the other five genetic strains. First instars of G20 and G30 responded similarly to dry heat treatments (Figure 1). First instars from G1 and G4 were comparatively more susceptible to increasing temperature than G7, G19, G20 and G30 (P < 0.001) (Figure 1). At 35 C, survival of G1 and G4 averaged 10% compared to 59% (range 51–65%) for G7, G19, G20 and G30 phylloxera (across combined exposure duration). At 40 C, survival of G1 and G4 averaged 4% compared to 40% (range 23–75%) for G7, G19, G20 and G30 at 75 min. Previous molecular studies by Umina et al. (2007) showed that G1 and G4 phylloxera are genetically similar and different from G7, G19, G20 and G30. These two phylloxera strains also differ in their susceptibility to disinfestation treatments. A recent study by Clarke et al. (2017a) showed that G1 and G4 phylloxera were most susceptible to sodium hypochlorite treatment compared to G7, G19, G20 © 2018 Australian Society of Viticulture and Oenology Inc. Clarke et al. Dry heat for grape phylloxera disinfestation 3 Figure 1. Survival of first instar phylloxera genetic strains (a) G1, (b) G4, (c) G7, (d) G19, (e) G20, and (f ) G30 when subjected to dry heat treatment at 22 ( ), 35 ( ), 40 ( ) and 45C ( ) for 75, 90 and 120 min. Table 1. Effect of dry heat on survival of first instars of four genetic strains (G7, G19, G20 and G30) on excised grapevine roots (Vitis vinifera cv. Chardonnay). Temperature of dry heat ( C) Total number of surviving first instars placed on grapevine roots First instars that established feeding sites (%) Pre-adult mortality (%) First instars that developed to adults that subsequently laid eggs (%) 82 38 35 89  2 35  3 18  7 <0.001 14 11  1 18  4 21  6 0.34 15 79  2 14  3 0 <0.001 6.5 22 35 40 P LSD (5%) LSD, least significant difference. and G30. Studies on the effectiveness of hot water treatment as a disinfestation method of diverse strains of grape phylloxera showed that G30 was most susceptible to hot water compared to G1, G4, G7, G19 and G20 (Clarke et al. 2017b). These previous studies supported by this current one highlight the importance of trialling disinfestation treatments across diverse phylloxera genetic strains. Limited effectiveness of some treatments pose as potential transfer risk of some strains between vineyards and quarantine zones. The finding that genetic strains vary in their susceptibility to temperature extremes may also have implications for quarantine risk under predicted climate change scenarios (Anderson et al. 2008). For G7, G19, G20 and G30 first instars that survived dry heat treatment at 22, 35 and 40 C, temperature had a significant effect on the proportion of insects that established feeding sites on V. vinifera and those that reached adulthood and reproduced (P < 0.001; Table 1). There were no significant differences, however, between genetic strains on establishment of feeding sites and reproduction after dry heat treatment (P = 0.550, data not shown). Of first instars that survived treatment at 35 and 40 C, 18 and 35% established feeding sites, respectively, compared to 89% of the Control (22 C) treatments (P < 0.01; Table 1), across the combined treatment durations and phylloxera genetic strains. Mortality of first instars on V. vinifera roots following treatment did not differ with temperature (P > 0.05; Table 1). First instars that developed into adults and reproduced averaged 14% (range 7–20%) at 35 C but none of those subjected to heat © 2018 Australian Society of Viticulture and Oenology Inc. treatment at 40 C produced eggs (Table 1). This finding implies that higher temperature impacts markedly on phylloxera development and reproduction. Overall, the results show that heat treatment of 35 C or lower does not inhibit phylloxera establishment, development and reproduction. Although there was no evidence to show that G20 and G30 phylloxera which survived at 40 C could reach adulthood and reproduce, dry heat at 40 C for 135 min is advisable as a precautionary measure to ensure 100% mortality across diverse genetic strains. Acknowledgements This work was funded by Wine Australia and Agriculture Victoria. The authors would like to acknowledge anonymous grapegrowers in north-east and central Victoria for allowing collection of insect and vine root material used in maintaining insect cultures. References Anderson, K., Findlay, C., Fuentes, S. and Tyerman, S. (2008) Garnaut climate change review: viticulture, wine and climate change (The University of Adelaide: Adelaide, SA) pp. 10–13. Buchanan, G.A. (1990) The distribution, biology and control of grape phylloxera, Daktulosphaira vitifolii (Fitch), in Victoria. PhD Thesis, La Trobe University, Bundoora, Vic., Australia. p. 179. Clarke, C.W., Wigg, F., Norng, S. and Powell, K.S. 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Manuscript received: 17 October 2017 Revised manuscript received: 12 December 2017 Accepted: 20 December 2017 © 2018 Australian Society of Viticulture and Oenology Inc.