Impacts of Soil Electrical Conductivity and Compost Mulches on Phylloxera Establishment and Abundance

Impacts of soil electrical conductivity and compost mulches on phylloxera establishment and abundance

Kevin Powell, Ginger Korosi, Bernadette Carmody and Rebecca Bruce

Department of Primary Industries, Rutherglen Centre, Biosciences Research Division, RMB 1145 Chiltern Valley Road, Rutherglen, Victoria 3685, Australia

Corresponding author: [email protected]

Introduction Grape phylloxera ( Daktulosphaira vitifoliae Fitch) is present in most grape-growing countries and can attack both the roots and foliage of grapevines. The root-galling form of phylloxera causes significant economic damage to Vitis vinifera L. and can also survive on the roots of ‘resistant’ rootstocks. In Australia grape phylloxera is a quarantine pest and is restricted to phylloxera infested zones in Victoria and New South Wales. Eighty-three genotypic clones of phylloxera have so far been identified in Australia (Umina et al. 2007) and some strains predominate in the different geographical regions. The degree of damage to the grapevine root system and the risk of phylloxera establishment through quarantine breakdown is predominantly influenced by the virulence of the phylloxera genetic strain (Powell 2008, 2011). Long-term management of root-galling strains of phylloxera has predominantly focused on recommending resistant rootstocks and developing robust quarantine protocols. This focus has largely ignored the impact that the soil environment, which is after all phylloxeras’ predominant habitat, has on the insect-host plant interactions. Future phylloxera management needs to consider a more integrated approach and where the influence of soil physiochemical properties on phylloxera establishment, subsequent abundance and the risk of spread are recognised. Newly developed options for targeted early detection of phylloxera are being developed, where soil factors are considered, include the use of a soil molecular DNA probe, trapping techniques and the use of soil electromagnetic induction surveys. Soil chemistry can also influence phylloxera establishment and abundance (Powell et al. 2003; Reisenzein et al. 2007) and their may be potential to modify these soil-pest interactions through soil management. Alternative approaches to phylloxera management using organic mulches may also be a potential option particularly when low virulent genetic strains of phylloxera are detected.

1 Phylloxera genetics and rootstock selection Different phylloxera strains can establish and develop colonies on resistant rootstocks to varying levels of adaptability and resistance-breaking biotypes have been reported on rootstock hybrids with partial V. vinifera parentage (Granett et al. 1985). However the genetic variability within phylloxera populations has been poorly understood until relatively recently. The development of improved molecular techniques over the last decade has seen an increased understanding and clarification of the life-cycle and genetics of grapevine phylloxera. Firstly with the development of random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) genetic markers (Fong et al. 1995; Forneck et al. 2000), and more recently with nuclear mitochondrial DNA markers (Corrie et al. 2003; Vorwerk and Forneck 2006). The complexity and range of genetic diversity characterised using these techniques has highlighted the potential for breakdown in phylloxera-resistant rootstocks and the need for selection of rootstocks based on phylloxera and grapevine genotypic background to reduce the risk of phylloxera transfer on machinery, grape products or footwear (Deretic et al. 2003; Dunstone et al. 2003; Korosi et al. 2009). The use of nuclear DNA microsatellite markers has resulted in the characterisation and geographical distribution of over 80 distinct phylloxera genotypes in Australia (Umina et al. 2007). Of these known genotypes most are root-feeding (radicicolae) only, but others are purely leaf-galling (gallicicole) and relatively few appear to be able to combine both a radicicolae and gallicicole life habitat (Corrie et al. 2003, 2004). Differing virulence levels of root-galling genetic strains on commercially-available rootstocks (Figure 1), novel hybirds and ungrafted V. vinifera have been reported (Korosi et al. 2007; Korosi et al. 2011a, b). This has important implications for management of ungrafted vines and selection of phylloxera- resistant rootstocks. In Australia when a new outbreak of phylloxera is detected insects are now routinely DNA typed and their population dynamics monitored in the field (Powell et al. 2003; Herbert et al. 2006). This assists the grower in determining both the choice of rootstock and timelines for a replanting program based on the insect genetics and population biology. DNA typing can also aid in traceback procedures to determine the origin of an infestation (Umina et al. 2007).

2 G1 Phylloxera survival

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Figure 1. Estimate of relative survival of two grapevine phylloxera genetic strains (a) G1 and (b) G19 on four rootstocks tested using an excised root bioassay system.

Quarantine and detection Grapevine phylloxera is a significant quarantine pest in Australia since its first detection in 1877 near Geelong, Victoria (Powell 2008). Quarantine protocols and regulations relating to the movement of grape material and vineyard machinery (NVHSC 2009) have been somewhat successful in minimising the risk of widespread distribution of phylloxera throughout all states in Australia. However, over the last decade a number of new infestations have been confirmed within the state of Victoria. The most recently created Phylloxera Infested Zone (PIZ) is the Maroondah PIZ, located in the Yarra Valley. Phylloxera was first detected there in December 2006, and at that time was thought to be contained to a single

3 vineyard until 2008-2010 when it was detected in a further 4 vineyards within the region resulting in an extension of the quarantine zone. This reaffirmed an important issue surrounding phylloxera detection; i.e. that an infestation may become established within a vineyard several years prior to physical visual evidence of vine stress. Early detection techniques are vital to contain the spread of phylloxera particularly highly virulent strains, such as G1 and G4 which survive and reproduce extremely well on ungrafted V. vinifera (Herbert et al. 2010). Some low virulence phylloxera genetic strains are also important and can be relatively difficult to detect because they cause less root damage and hence visual symptoms above ground are less apparent. Early detection of all phylloxera strains is essential to ensure the sustainability and profitability of the viticulture industry in Australia. Late detections can result in insignificant economic cost to affected growers, with replanting onto phylloxera resistant rootstocks costing up to AU$25,000 per hectare and the additional costs associated with implementation of quarantine protocols.

Conventional detection The first stage in any phylloxera management program is to detect the insect, which is a challenge due to its small size and seemingly random spatial distribution. A range of detection options have been developed (Renzullo et al. 2004; Herbert et al. 2007; Powell 2007a). The root-galling form spends most of its life below-ground phylloxera feeding on the grapevine root system, although in spring and summer it does emerge from onto the soil surface causing increased quarantine risks (Powell et al. 2000). It can be present in high abundance or in relatively low numbers depending on its genetic identity, its host plant genotype, climatic conditions and the soil environment. Generally the first indication that phylloxera may be present in a vineyard is shown by show stress symptoms in the foliage or canopy. This can be expressed as premature senescence in autumn, stunting of lateral shoot growth, reduced grape yields, reduced overall vigour or a general weak spot within a group of vines. However, once a weak spot is detected phylloxera has been present for several years and is highly likely to have spread to other vines which appear seemingly vigorous and asymptomatic. Conventional detection methods rely on systematic vine root surveys to inspect for the physical presence of phylloxera, but this technique is costly due its labour intensive nature. Remote sensing systems to generate multispectral imagery for identification of areas of reduced photosynthetically-active biomass on both a vineyard- and area-wide scale (Renzullo et al. 2004; Frazier et al. 2004) have also been used. While remote sensing is successful in identifying areas of poor canopy vigour for follow-up ground survey, these ‘weak spots’ may be caused by any number of non-specific stresses including water or nutrient stress and unrelated pest and diseases (Renzullo et al. 2004). Healthy canopy vigour,

4 particularly in wet seasons, may also disguise the expression of above-ground signs of root degradation, particularly if the infesting phylloxera genotype is either of low virulence, relatively unsuited to the environmental conditions, or if the vineyard is planted with tolerant rootstock varieties (Korosi et al. 2007; Herbert et al. 2008).

Emerging detection techniques Three primary (i.e. where either the insect or its DNA are examined directly) techniques are available for phylloxera detection. Roots surveying is the conventional primary detection method and two emerging non-destructive sampling technologies are currently being trialled, emergence traps and soil molecular probes, which may offer greater specificity for phylloxera detection. Emergence traps placed on the soil surface to capture a sample of the emerging phylloxera population. Emergence traps are best used in the spring and summer months to coincide with a peak in emerging phylloxera populations (Powell et al. 2009). A soil-based assay using a phylloxera-specific DNA probe which directly detects the presence of phylloxera DNA (Herbert et al. 2004, 2008) is also applicable. These two techniques were initially trialled over a single season in the Yarra Valley, Victoria in 2006-2007 and showed improved sensitivity when compared with visual root observations (Herbert et al. 2008). However even though both emergence traps and DNA probe appear to offer greater sensitivity than conventional surveys, without a way to target the sampling required with these techniques, the problems surrounding labour requirements and timing for optimum detection throughout the year remain. There is a need to be able to consider how soil factors may influence spatial distribution of phylloxera and hence detection efficiency in a way that may allow primary detection techniques, such as DNA probes and emergence traps, to be targeted to those areas that have a higher risk of phylloxera establishment. To facilitate the development of a targeted detection approach studies have recently been conducted in the Yarra Valley which compare the efficiency of monitoring techniques by coming with a secondary detection approach using soil mapping. The combined approach includes mapping of soil apparent electrical conductivity (EC a) derived using an electromagnetic induction soil sensor, and direct measures of phylloxera incidence using phylloxera-specific DNA analysis, visual root surveying and phylloxera emergence trapping.

Soil sensing A three-year pilot study (2009-2011) comparing the efficiency of phylloxera detection systems (emergence traps, visual root observation and soil DNA probe) and developing a targeted detection strategy based on soil properties (using EM38) was conducted in the Yarra Valley, Victoria (Bruce et al., 2011).

5 Soil EC a was measured using an electromagnetic induction sensor (EM38, Geonics, Ontario, Canada) across two vineyard blocks, to measure the apparent electrical conductivity

(mS/m) of the soil. Soil EC a is determined by the relative amount and types of clay, salts, rock and moisture in the soil profile (Proffitt et al. 2006). The EM38 sensor was operated in vertical dipole mode, which is the most effective at capturing soil information from 10-150 cm, mounted on a rubber sled and towed behind an all-terrain vehicle. The continuous output data was captured using a Trimble TSCe® data-logger along with geo-location data provided from a Trimble differential global positioning system (DGPS). Ground surveys were conducted by visual root inspection with a hand lens in every 5th panel of every 3 rd row to confirm the presence of phylloxera life-stages. A phylloxera-specific, soil-based DNA assay (Herbert et al. 2008) was used to confirm infestation points within the two infested blocks. 2.5 cm diameter soil cores to a depth of 20 cm were taken from within a 30 cm radius of the trunk of vines Soil samples were transported back to DPI Rutherglen under quarantine conditions and oven dried at 50°C for 48 hours to devitalise the phylloxera prior to transport to SARDI, South Australia. To allow comparison of the relative effectiveness of phylloxera emergence traps, root surveys and soil-based phylloxera DNA detection, a grid sampling pattern following the established protocol for ground surveys was established.

Both blocks showed variation in EC a, with clearly defined areas of relatively low to high conductivity. Grid sampling detailed the distribution of the phylloxera infestation throughout both blocks (Figure 2 shows example of one block). Phylloxera was found across a range of soil EC a levels, with higher numbers occurring in the mid-range. This is potentially indicative of a preference of phylloxera to establish in higher numbers in areas of relatively mid to high EC a, with subsequent spread throughout the blocks due to natural dispersal, on machinery or on vineyard workers. This follows a trend seen at previous sites

(Bruce et al. 2009), where phylloxera was initially detected in areas of high EC a in larger numbers, then moving over time to lower EC a areas. Establishment was inferred from state of vine health and the total phylloxera numbers found in emergence traps from regular population monitoring. A comparison of the number of vines with confirmed phylloxera infestation (Figure 2) showed that the root survey method, soil-based DNA detection method and the emergence trapping method all produced similar results. The emergence trapping was marginally more sensitive to phylloxera presence.

8.8 ms/m 69 ms/m

Figure 2. An electromagnetic induction (EM38) survey of a study block in the Yarra Valley. A comparison of the relative effectiveness of ground survey (March 2010), soil-based DNA testing (March 2010) and emergence trapping (January –February 2010) is shown. Open circles indicate no phylloxera was found. Filled circles indicate phylloxera was found.

Alternative management Although there have been some reports of potential breakdown in rootstock resistance to phylloxera in Europe (Porten et al. 2000) and the USA (Granett et al. 2001) these are limited. However, interim or alternative management options for phylloxera require some consideration. One option is the use of composted organic mulches. In temperate horticultural systems compost applications can have a number of beneficial effects including pest suppression (Brown and Tworkoski 2004), enhancement of beneficial organisms (Mathews et al. 2004) and increased plant vigour (Pinamonti 1998). Composts in a variety of formulations have been reported to suppress plant pests and diseases either directly, through chemical or physical interactions, or indirectly by affecting the host- plant physiology (Akhtar and Mahmood 1997). Mulch application can change the physical and textural properties of the soil environment making it either more or less conducive to phylloxera feeding on the root system or directly affect the insects’ mobility through the soil. Moisture content and temperature of the soil could also vary under differing soil management conditions. Phylloxera development is directly affected by vine physiology (Omer et al. 2002), moisture and temperature changes in the environment (Granett and Timper 1987). The chemical composition of a mulch formulation is likely to impact on grapevine physiological response and hence phylloxera- grapevine interactions. Only two studies have been conducted in Australia to assess the impact of mulches on phylloxera populations and these focused on the use of composted green waste and composted marc (Powell et al. 2007b, c).

Composted green waste study Trials were conducted in a commercial vineyard, located within the King Valley Phylloxera Infested Zone (PIZ), North-East Victoria, Australia. Phylloxera was first detected in the vineyard in May 1997. The phylloxera genetic strain at the site was characterised as G4. The trial was conducted between October and June over three consecutive growing seasons. The commercial vineyard block chosen for the study consisted of 15-year old ungrafted Vitis vinifera L. ‘Sauvignon Blanc’ vines, which allowed the comparison of composted treated and untreated phylloxera-infested grapevines under field conditions. Mature composted green waste was prepared commercially through a windrowing process following Australian composting standards (Anon. 2000) and analysed prior to application. Treatments were applied as composted green waste in October, at depth of 5cm.

8 Four rows of vines (2 sets of adjacent rows with a buffer row between) were designated as compost trial blocks. Along the rows there were alternating experimental blocks of compost and control (ie no compost) treatments. In each block there were 10 vines. In each plot two central grapevines were used as ‘population monitor vines’, to assess above- and below-ground abundance of phylloxera life stages. To assess phylloxera population movement aboveground emergence traps (Powell et al. 2000), and trunk traps (data not presented) were used fortnightly for each monitor vine.

All plot-level data were analysed using either standard or repeated measures Analysis of Variance (ANOVA). Data collected over time (emergence traps) were analysed using repeated measures ANOVA. Data on insect abundance from emergence trapping were log 10 transformed prior to analysis in order to satisfy the assumptions underlying the ANOVA procedure. Fisher’s unprotected least significant differences (LSD at 5%) were used to calculate significant differences between treatments.

In the first season crawler counts in emergence traps peaked between early January and early February (Figure 3). Over the entire sampling period significantly higher (p<0.001) numbers of first instars were recorded from the composted vines compared with the control vines. On three consecutive sampling dates the difference between treatments was significant (p<0.05) (Figure 3a). In the second season the overall abundance of phylloxera first instars (Figure 3b) was lower than the first season (Figure 3b). Significantly more first instars were collected in the traps of compost treated vines than control vines (p<0.001) over the whole sampling period. On four consecutive sampling dates significant differences between treatments were recorded (p<0.05). In the third season (data not presented) there was no overall significant effect of treatment on crawler number (p<0.05).

Figure 3. Seasonal first instar phylloxera abundance above-ground (mean per emergence trap) of composted and non-composted Vitis vinifera ‘Sauvignon Blanc’ during (a) first and (b) second growing seasons after mulch application. Asterisks denote significant differences between crawler abundance in traps (*P<0.05).

Composted marc study Trials were conducted in a commercial vineyard, with winery on-site, located within the Nagambie PIZ, Central Victoria, Australia. Phylloxera was first detected at the property in 1974. The experimental block consisted of twelve split rows of ungrafted V. vinifera L. ‘Semillion’ grapevines planted. The phylloxera genetic strain at the site was characterised as G1. Mature composted marc was prepared on-site using a windrowing process (Anon. 2000). Three compost formulations were used as treatments and designated as M1 (a mixture of composted marc and pig litter (3:1 ratio)), M2 (a mixture of composted marc and wood shavings and (3:1 ratio)) and M3 (100% composted marc). The experiment was laid out in a nested randomised complete block design with 6 blocks. In each block, there were 4 plots (one for each treatment and the control). In each of three compost treatment plots, a single compost formulation was applied to a depth of 20cm at the commencement of the experiment in October just prior to budburst. The control plots had no compost applied to the soil surface. In each plot two adjacent vines for above-ground population monitoring were selected at which a single plastic emergence trap was placed adjacent to the vine trunk to monitor phylloxera first instar dispersal (as described in Powell et al. 2000) at fortnightly intervals. To assess population numbers below-ground a destructive root sampling technique was used twice per growing season in winter and late summer (data not presented). All plot-level data were analysed using either standard or repeated measures Analysis of Variance (ANOVA). Data collected over time (emergence traps) were analysed using repeated measures ANOVA. Data on insect abundance from emergence trapping were log 10 transformed prior to analysis in order to satisfy the assumptions underlying the ANOVA procedure. Fisher’s unprotected least significant differences (LSD at 5%) were used to calculate significant differences between treatments. Following application of mulches phylloxera first instar (crawler) emergence above- ground was monitored above ground using emergence traps from November to March for three consecutive seasons. In the first season crawlers were more abundant from December and March and peaked in early January on the treatment and control vines (Figure 4a). There were significant differences crawler abundance between counting periods (p<0.001) with higher numbers recorded during the summer period (January-February). On two consecutive sampling dates (14 th December and 28 th December) the M1 and M2 compost treatments showed significantly lower (P <0.05) emergence levels than both the non-compost and the M3 compost treatment (Figure 4a). On one sampling date, later in the season, the non-compost

11 treatment had significantly lower levels than the M1, M2 and M3 treatments. Overall there was a significant interaction between treatment and time (P<0.05). In the second season overall crawler abundance (Figure 4b) was lower than the first season (Figure 4a). There were significant differences in crawler abundance between counting periods (P<0.001) with higher numbers recorded during the summer period (January- February). With the exception of the control, which showed an additional emergence peak in spring, this was the case for all compost treatments (M1, M2 and M3). On two consecutive sampling dates (28 th November and 10 th December) significant effects were recorded (P <0.05) with the no-compost treatment having higher phylloxera emergence levels than all three compost treatments. On one sampling date (17 th January) the M2 treated vines had significantly higher phylloxera emergence levels than the M1, M3 and no-compost treatments. In the third season overall the abundance of phylloxera crawlers (Figure 4c) was higher than the previous season (Figure 4b). Over the entire sampling period, in the third season after treatment application, significant differences were observed between the log number of crawlers in the composts (M1, M2 and M3) and the control (P<0.05). There were significant differences in crawler abundance between the counting periods (P<0.001). The highest numbers of crawlers caught in the emergence traps was during the summer months (January-February). On three consecutive sampling dates (14 th January, 28 th January and 11 h February) significant effects were recorded (p <0.05) with the M1 and M2 treatments having lower phylloxera emergence levels than both the M3 and the no-compost treatments. Overall there was a significant interaction between treatment and time (P<0.05).

Figure 4. Seasonal first instar phylloxera abundance above-ground (mean per emergence trap) of composted (M1, M2 and M3) and non-composted Vitis vinifera ‘Semillion’ during (a) first (b) second and (c) third growing seasons after mulch application. Asterisks denote significant differences between crawler abundance in traps (*P<0.05).

Conclusions and future directions Root-feeding phylloxera genetic strains predominate in phylloxera-infested vineyards in Australia and some of these strains cause significant economic damage to ungrafted V. vinifera grapevines. Despite the fact that these genetic strains spend a large proportion of there life-cycle below-ground in a soil environment feeding on the grapevine root system, surprisingly little is known about the impact that soil chemical and physical properties have on the insects ability to locate its host, establish and subsequently develop. Preliminary results from early detection trials in the Yarra Valley indicate relationships between soil properties and phylloxera spatial distribution and further studies are required to fully elucidate the mechanism which influences phylloxera establishment and dispersal under different soil conditions. It is certain that soil risk-mapping, when linked to improved early detection protocols, could provide in the future a more effective way of detecting phylloxera early and thereby improve the possibilities of restricting its spread and reducing the economic damage it causes. Earlier and more targeted detection methods for grapevine phylloxera will ultimately lead to early intervention and improved management of this destructive insect pest. Future research directions need to focus on (i) a more comprehensive understanding of how the genetic diversity of phylloxera influences its interactions with the host plant ( Vitis species) at the root zone level (ii) improving our understanding of how soil properties can influence establishment and dispersal of different phylloxera strains and (iii) an integrated approach to management. Alternative short-term phylloxera management approaches, where the soil environment is modified, have been considered in some trials in Europe and Australia but the future use of organic mulch formulations for phylloxera suppression will require further study before they can be used as part of an integrated phylloxera management program. In the two Australian studies conducted so far it is apparent that formulation is a key parameter when determining the effectiveness of any compost mulch application. Some composted marc formulations when applied as mulch, can be a beneficial management option in phylloxera- infested vineyards by reducing above-ground dispersive stages of the population at certain times of the year, whilst other formulations such as composted green waste may increase phylloxera emergence levels and hence the quarantine risk. These observations emphasise the need for studies where a range of compost formulations are studied to quantify their relative effectiveness.

Acknowledgements

14 The authors would like to thank the ASVO and The Phylloxera and Grape Industry Board of South Australia for their support. The research presented was financially supported by Grape and Wine Research and Development Corporation, The Phylloxera and Grape Industry Board of South Australia and the Department of Primary Industries, Victoria.

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