Status of thousand cankers disease on eastern black walnut in the eastern United States at two locations over 3 years

For. Path. 45 (2015) 203 214 doi: 10.1111/efp.12154 2014 The Author. Forest Pathology Published by Blackwell Verlag GmbH This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Status of thousand cankers disease on eastern black walnut in the eastern United States at two locations over 3 years By G. J. Griffin Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061, USA; E-mail: gagriffi@vt.edu (for correspondence) Summary Thousand cankers disease (TCD), a lethal fungal dieback of eastern black walnut (Juglans nigra), caused by Geosmithia morbida, and spread by the walnut twig beetle, Pityophthorus juglandis, was documented in 2009 to be very destructive in the western United States and was identified in the native range of J. nigra at Knoxville, Tennessee, in 2010, and in 2011 at Richmond, Virginia. Beginning late 2010, we studied branch dieback levels (per cent live crown) and new TCD symptom development at these two quarantined locations monthly for 3 years. Of the 106 trees studied (53 at each location), 31 trees had low live crown ratings of 70 to 0% with little change over the 3 years of the study. One per cent of the trees developed new symptoms on a per-year basis. Thus, a moderate level of TCD (mean = 76% live crown) was present in these two locations, and most trees were in a quiescent or dormant TCD condition for 3 years, an important finding not previously reported. We found new TCD symptoms developed in Richmond in 2011 and 2012 when precipitation from January 1 to the end of August was low (60 64 cm), and not when the precipitation in Richmond was higher (99 cm). In late 2012, in Richmond, soil water potential assays indicated that some black walnut trees were under severe physiological stress ( 15 bars). In contrast, in 2013, high precipitation levels (99 and 130 cm) and high soil water potentials ( 0.1 to 3 bars) at both locations were associated with extensive new foliage and stem growth and recovery from TCD. Further research is needed on water relationships in regard to TCD and black walnut health. 1 Introduction In the western United States, decline and mortality of eastern black walnut (Juglans nigra L.) trees were noted as early as 2001 (Tisserat et al. 2009). Affected trees exhibited leaf yellowing and thinning in the upper crown, which was followed by branch dieback. After initial symptoms, the black walnuts were killed typically within 2 years. Over 700 trees were killed and removed in Boulder, CO alone by 2008, which represented the majority of black walnut trees there. Evidence was provided that the black walnut mortality was associated with the walnut twig beetle, Pityophthorus juglandis Blackman and the fungus, Geosmithia morbida Kolarik; Fusarium solani (Mart.) Sacc. contributed to disease development in the late stages (Tisserat et al. 2009, 2011). The disease was called thousand cankers disease (TCD) because of the large number of cankers that developed in phloem tissues beneath the bark surface. The beetle is native to the south-western region of North America, where the beetle, and presumably G. morbida, is found on J. major (Torr.) A. Heller (Tisserat et al. 2011; Utley et al. 2013). The authors indicated that the disease posed a serious threat to J. nigra in the natural range of the species in the eastern United States. In addition, black walnut export logs may spread TCD to other countries. In 2010, TCD was identified in the eastern United States in Knoxville, Tennessee (http://protecttnforests.org/thousand_canker.html), and in 2011 in the area of Richmond, Virginia by Norm Dart of the Virginia Department of Agriculture and Consumer Services (VDACS) (www.vdacs.virginia.gov/plant&pest/disease-tcd.shtml), which was confirmed by the Virginia Tech Plant Disease Clinic (Hansen et al. 2011). In 2011, TCD was also found in Bucks County, Pennsylvania (www. pabulletin.com/secure/data/vol41/41-35/1462.html). Cankers and dieback in the East were similar to that observed in the West, and both the walnut twig beetle and G. morbida were associated with the disease in the East. Both research areas in this study, Knoxville and Richmond, are now quarantined, which limits the movement of black walnut in and out of the quarantined areas. Eastern black walnut is a mesic (moist site) tree species that is shade intolerant, but has a widespread distribution in forests of the eastern United States, ranging from parts of New England and southern Minnesota in the North to south-eastern Texas and upper Florida in the South (Fowells 1965). Often black walnut is an infrequent component of the forest tree population where is it is found in natural forest stands (Braun 1950). One exception is on some alluvial soils along rivers in the Appalachian Mountains where it can be a dominant species and obtain a large height and diameter. Also, it is one of four dominant species and has obtained large size in areas of Indiana in the western mesophytic forest region (Braun 1950). In Indiana, black walnut trees were 46 m tall and 1.8 m in diameter on the most favourable sites (Fowells 1965). It grows best on deep, well-drained nearly neutral soils which are moist and fertile. Eastern urban areas and plantations where black walnut has been planted may or may not be representative, however, of the environmental conditions found at the better natural sites for black walnut growth and vigour. In addition, construction projects, especially involving excavations near roads or during building construction, may have altered the natural soil and topographic characteristics in urban landscapes to a condition unfavourable to black walnut growth and vigour. This study was undertaken to evaluate over 3 years (2011 2013) the status of TCD on individual eastern black walnut trees in Knoxville, TN, and Richmond, VA, and to evaluate, as much as possible, in these TCD-quarantined areas, environ- Received: 21.7.2014; accepted: 24.9.2014; editor: S. Woodward http://wileyonlinelibrary.com/

204 G. J. Griffin mental factors that may be associated with TCD development. As black walnut is a mesic tree species, special attention was given to rainfall (precipitation) amounts and soil water potential values in the black walnut feeder root zone found at these two locations. In addition, concentrations of the amino acid proline in black walnut leaf tissues were evaluated during the course of the study. Proline has been identified as an amino acid associated with abiotic stress responses in some plants (Orcutt and Nilsen 2000). The non-tcd area of Blacksburg, VA was used as a reference area for this study with evaluations performed as were performed in the two TCD areas. This is the first report of TCD status over time in the eastern United States. 2 Materials and methods 2.1 Eastern black walnut trees studied in the TCD areas of Knoxville and Richmond and in the non-tcd area of Blacksburg In Knoxville, beginning in September, 2010, near the location where TCD was first identified (the TCD-founder site) in the eastern United States, we evaluated TCD development on 53 individual black walnut trees over 3 years. Initially, a greater number of trees were evaluated, but some trees were removed for forestry research studies, by storm damage and others by land owners. Most (29) black walnut trees at the first location, A, were established landscape trees, except for six canopy black walnut trees growing in mixed hardwood forest stands adjacent to the landscape areas. In addition, at a second location, B, about 2 km distant from the first location, 14 established landscape black walnut trees, two canopy black walnut trees in adjacent mixed hardwood forest stands and two small, volunteer black walnuts on the edge of a mixed forest stand were evaluated for TCD development. Both locations were near the centre of the TCD quarantine area and included trees exhibiting severe to slight dieback at the start of the study. One black walnut tree at the TCDfounder site in September, 2010 had no live branches but had live epicormic sprouts at the tree base, suggesting it was diseased recently. In the Richmond area, south of the city, beginning in early July 2011, soon after identification of TCD on two black walnut trees (the TCD-founder site) in late June by Norm Dart (VDACS), we evaluated TCD development on a small number (11) of black walnut trees near the TCD-founder site. At the same time, Norm Dart, stationed in Richmond, established a TCD quarantine area for Richmond (www.vdacs.virginia.gov/plant&pest/disease-tcd.shtml). In 2012, additional black walnut locations, identified by Norm Dart for the quarantine survey, were included in our TCD development studies so that 53 trees were evaluated for most of 3 years. Some (18) of the black walnut trees grew in a mixed hardwood forest stand adjacent to landscape black walnut trees at one of the locations. There were three locations overall: one south, one west and one north of the urban area of Richmond. Initially, a greater number of trees were evaluated, but as found in Knoxville, some trees were removed during the course of the studies before 2013, some in association with constructions projects, one by a land owner because of dead hazardous branches and one because of storm damage. All black walnuts evaluated were within the Richmond TCD quarantine area, established by VDACS and exhibited severe to slight dieback. The non-tcd area of Blacksburg, VA was used as a reference area for this study with evaluations done as were done in the two TCD areas. In the non-tcd area of Blacksburg, both landscape and natural forest black walnut trees were evaluated for 3 years in the same manner as in Knoxville and Richmond. In this area, 30 landscape trees were evaluated in one location and 25 canopy natural forest trees growing on alluvial soil adjacent to a mountain river were evaluated in a second location, for a total of 55 trees evaluated for 3 years. All but two small trees in the three geographic areas were established landscape or canopy forest trees. The diameters of the landscape and forest trees studied in Knoxville, Richmond and Blacksburg were generally similar and had diameters of 26 113 cm at breast height (dbh), except for the two smaller volunteer black walnut trees in Knoxville at the edge of a forest stand, which were 15 and 8 cm dbh. 2.2 Evaluation of TCD at monthly intervals To evaluate TCD, we made monthly live crown evaluations on each tree visually: a) by examining trees for TCD symptoms and b) by estimating the percentage of the tree crown with live foliage, to the nearest 10%, on the study trees each year, beginning in May and ending in late August or early September. In some years, additional evaluations were made to assess symptom development. Evaluations were also initially performed in April, but due to the variation among individual black walnut trees in the timing of bud break in April, good evaluations for all study trees could not be made during this month. Earlier extensive evaluations in several non-tcd areas indicated that most landscape black walnut trees had small amounts of dead branchlets and branches amounting to 0 10% of the canopy with a mean of about 5%. This may be due to wind damage and other causes. Only young, small trees exhibited no evidence of dead branchlets or branches and only two small trees were included in this study, as mentioned. Photographs were made almost monthly of representative black walnut tree crowns in Knoxville, Richmond and Blacksburg. If, during the course of the study, a tree exhibited new TCD symptoms, such as leaflet yellowing in the upper tree crown, wilting, leaflet drop, dead clinging leaves or dieback, the amount (per cent of the existing green foliage) was recorded and photographed. 2.3 Soil properties and collection of soil samples for soil water potential analyses In Knoxville, two representative black walnut tree sites in the landscape locations, adjacent to the forest locations, were selected for evaluating soil properties and monthly soil water potential evaluations from 2012 through 2013. In Richmond

Status of thousand cankers disease 205 Table 1. Soil physical and chemical properties at eastern black walnut research sites affecting black walnut tree vigor and health status in TCD and non-tcd areas. Soil/Tree code Soil texture Available water 1 (%) Organic matter (%) ph Ca kg/ha Mg kg/ha P kg/ha K kg/ha Knoxville (TCD) KLST6 Silt loam 12 5.8 7.6 5176 466 58 357 KLST7 Silty clay loam 10 5.2 7.4 8244 664 13 208 Richmond (TCD) RCHT1 Loam 14 4.2 5.9 1254 146 7 150 RCST2 Sandy loam 8 3.3 6.7 1987 254 21 176 Blacksburg (non-tcd) BVT2 Silt loam 16 5.6 6.7 3219 506 31 316 BNRT1 Loamy sand 10 2.4 7.9 3531 823 28 102 1 Equals the difference between % water at -15 bars soil water potential (no available water) and 0.3 bars (field capacity) for soil samples collected in the 0 25 cm soil depth region (feeder root zone) under the tree canopy. Expressed as % water on an oven-dry soil weight basis. and Blacksburg, one site for both was in a landscape location, one site was adjacent to a forest stand (Richmond), and one site (Blacksburg) was in the natural forest alluvial soil location. At all three areas, soil samples at the start of this investigation, and later monthly samples during live crown evaluations, were collected under the tree canopies with a soil auger to a depth of approximately 25 cm for soil chemical and physical characterization. Soil samples were placed in zip-lock plastic bags for transport to Virginia Tech in a cooler for physical and chemical analyses. Soil texture (per cent sand, silt and clay) was determined for each soil in the Virginia Tech Soil Physics Laboratory as were soil water potential properties. The latter were determined by the pressure-plate technique at values ranging from field capacity ( 0.3 bars matric potential) to the critical moisture point ( 15 bars matric potential). The difference in soil water content (per cent water) between these two values indicates the amount of water available to plants for growth. A soil water retention curve of per cent water versus matric water potential values, determined in the pressure-plate assays, was drawn from the data. Per cent soil water content was determined gravimetrically by drying field soil samples at 105C for 24 h. Chemical analyses for soil Ca, Mg, K, P, Fe, soluble salts, ph and per cent organic matter were determined in the Virginia Tech Soil Testing Laboratory. The results of these assays are indicated in Table 1. All soils had generally satisfactory chemical and physical properties except for the sandy loam soil, RCST2, with the lowest available soil water property and RCHT1 soil having low P. Assay of soil water potential at the two sites in each area was performed monthly each year at the time per cent live crown evaluations were made. Soil samples were collected and assayed as indicated above. For soil water potential determinations at each site for each month, the per cent water content for each field-collected sample was compared to the soil water retention curve drawn from the data found in the initial soil pressure-plate assays, described above. Precipitation amounts for the Knoxville, Richmond and Blacksburg areas were obtained from records of the National Oceanographic and Atmospheric Administration (NOAA). 2.4 Collection of leaves for monthly proline analyses Each month at either Knoxville, Richmond or Blacksburg, when per cent live crowns were evaluated, two representative leaves, each containing many leaflets, were randomly collected from each tree with a tree pruner. The same trees used for soil water potential evaluations were used for the proline assay. Black walnut leaves were collected, the leaflets separated from the leaf rachis, and the leaflets cut into small pieces before placing them in a zip-lock plastic bag. The bag containing the leaf samples was immediately placed in a portable freezer at 15 C for transport to the laboratory for analysis. Proline concentrations on a fresh weight basis were determined in the Virginia Tech laboratory by the method of Carillo and Gibon (2011) with three replications per assay. 3 Results 3.1 Status of TCD in Knoxville and Richmond: per cent live crown (branch dieback) evaluations, 2011 2013 Per cent live crown evaluations yielded information on the amount of branch dieback on each black walnut tree and thus the severity of TCD. In this study, a rating of 80 90% live crown was considered slight dieback, a rating of 60 70% live crown was considered moderate dieback, and a rating of 50 to 0% live crown was considered severe dieback (Table 2). Overall, 31 of 106 trees in 2012, and 30 of 105 trees in 2013, had live crown ratings of 70 to 0% (Table 2). One tree (30% live crown), with new TCD, was cut down in 2012, reducing the number of trees from 106 to 105 in 2013. Ratings in Knoxville and Richmond were somewhat similar (Table 2). Per cent live crown ratings on most black walnut trees decreased very little from 2011 to 2013, except for a few trees developing new TCD symptoms (see below), suggesting overall that TCD was in a quiescent or dormant state. Some trees with moderate dieback (Table 2) had improved live crown conditions due in part to new stem and leaf growth and fallen dead branches (see below).

206 G. J. Griffin Table 2. Per cent live crown ratings in 2013 for eastern black walnut trees evaluated monthly from May until early September in the TCD areas of Knoxville, TN and Richmond, VA, and in the non-tcd area of Blacksburg, VA 1. Knoxville area (TCD) Richmond area (TCD) Blacksburg area (non-tcd) % Live crown No. of trees % Live crown No. of trees % % Live crown No. of trees 90 100 Slight dieback 25 90 Slight dieback 21 90 Slight dieback 50 80 14 80 15 80 4 70 Moderate dieback 5 70 Moderate dieback 4 70 Moderate dieback 0 60 2 60 2 60 0 50 Severe dieback 6 50 Severe dieback 1 50 Severe dieback 0 40 2 1 40 6 40 0 30 0 30 0 30 0 20 0 20 1 20 1 3 10 0 10 2 1 10 0 0 0 0 4 1 0 0 Total 53 5 52 6 55 1 No new TCD symptoms or decreases in per cent live crown ratings were observed at either Knoxville or Richmond during 2013. Last 2013 live crown evaluations were done in late August and early September. 2 Two trees (40 2, and 10 2 above) in Knoxville and Richmond were removed during 2013, due to a hazardous branch condition following May and June, 2013, live crown evaluations, respectively. 3 Most of the crown of one black walnut tree (90% rating) was destroyed in 2012 by a severe storm in the natural forest stand on alluvial soil. 4 This tree was still alive at the end of 2012, but not in 2013. 5 Other black walnut trees were removed at the Knoxville research sites during 2011 and 2012 due to use by foresters for research, by storm damage, or by land owners. 6 Other black walnut trees were removed at the Richmond research sites during 2011 and 2012 due to construction projects, storm damage, or by land owners. One of 53 trees studied developed new TCD symptoms in 2012 and was removed before 2013 by the owner, leaving 52 trees at the start of 2013. 3.2 New TCD symptom development patterns and other changes on black walnut trees The first new TCD symptoms observed on black walnut trees in the two study areas was on 5 July, 2011 in the south Richmond location on TCD-founder tree, RST2, shortly after TCD was found in the Richmond area on this tree and an adjacent tree in late June by Norm Dart (VDACS). Both trees had severe TCD (extensive dieback) with tree RST2 having a 20% live crown rating plus several epicormic sprouts on the main stem. The live portion of RST2 was a large, long, scaffolding branch that extended away from the main stem into full sun. The new symptoms were in a small area of foliage on a branchlet that exhibited off-colour bronzing and incipient wilt (Fig. 1, top). When this tree was examined about 1 month later on August 2, this same branchlet area had no foliage and, in addition, new adjacent small areas on branchlets exhibited symptoms similar to those observed on July 5. On 2 September 2011 all leaves on branchlets that were symptomatic on 2 August 2011 were dropped and no new foliage symptoms were apparent (Fig. 1, middle). After this date, no new symptoms were observed on this branch during 2012 or in 2013. In fact, the remaining foliage on the branch became more dense by June 6, 2013 (Fig. 1, bottom). After 6 June 2013, the tree was cut back to the main trunk by the owner because of hazardous, previously dead branches on the tree. For the eastern black walnut population studied, we termed this Type 1 TCD symptom sequence or slowed and quiescent dieback on branchlets. A second type of TCD development was observed in 2012 in the Richmond area on May 4. One black walnut tree north of the Richmond urban area exhibited green foliage wilt in two parts of the canopy (Fig. 2, top). This tree had severe TCD at this time with a 40% live crown rating. About 15% of the existing or remaining green foliage exhibited the green foliage wilt. On June 4, the tree had dead, clinging leaves (Fig. 2, bottom) in the same area of the tree where green foliage wilt was observed. On July 2, the tree had a 30% live crown rating. After this date, the owner removed the tree from his property. We term this Type 2 TCD symptom sequence, or green foliage wilt/dead clinging leaves, for the population of black walnut trees we studied. No leaflet yellowing was observed during 2012 on this tree. Very similar symptoms to Type 2 were observed by Norm Dart (VDACS) in the Richmond area at another location and at about the same time (personal communication). Later in the growing season in 2012, at a site very close (about 55 m distant) to the tree with the Type 2 TCD symptoms, we observed a third type of TCD symptom development. This tree had an 80% live crown rating on May 4, June 4 and July 2, a 60% rating on August 1 and a 40% rating on Sept. 6. On the August 1 date, extensive yellowing of leaflets, leaflet drop, incipient wilt and branch death in the upper tree crown were observed (Fig. 3, top and bottom). A few nuts had formed, but no nuts on the branches were dropped. A few dead, clinging leaves were observed on this tree on 6 September 2012. On May 6, 2013 the tree was dead. We call this Type 3 symptom sequence or upper tree crown yellowing/leaflet drop. This symptom sequence is similar to the symptom development that has been observed in the West. Throughout the 3-year study, we observed for many trees, especially in Knoxville during 2012 and 2013, that the foliage became more dense and extensive if no new TCD symptoms developed on the tree. This growth was observed even for trees with as low as a 40% live crown rating. However, this occurred only in areas of the tree that had at least some rem-

Status of thousand cankers disease 207 Fig. 1. Type 1 TCD symptom sequence (slowed and quiescent dieback) observed in the south Richmond, VA TCD area on branchlets of TCDfounder tree, RST2, with previous severe TCD progression. (Top photograph) 5 July 2011. A small area of foliage on the upper, central portion of a branchlet of a large horizontal branch exhibited leaf bronzing and incipient wilt (arrow) on this date, and all leaves were dropped from this small area by 2 August 2011. Some adjacent new bronzing symptoms were also present on August 2. (Middle photograph) 2 September 2011. Photograph shows the absence of leaves in areas mentioned above. Only slight progress of TCD symptom development (leaf drop) occurred after this date. No new leaflet bronzing, yellowing, wilt or dead leaves were observed in 2012 on this tree. (Bottom photograph) 6 June 2013. No TCD progress was observed, and growth of healthy, green foliage on this branch occurred during 2013. nants of living foliage on portions of the branches. Branches or branch parts that appeared completely dead and were not dropped did not develop this new foliage. At the end of the growing season in 2012 and 2013, a few trees appeared remarkably healthy, such as tree KLST1 in Knoxville. This tree had a 60% live crown rating on 14 September 2010 but was given a 90% live crown rating in 2012 and 2013 (Fig. 4). Part of this healthy appearance was due to the dropping of some dead branches and part was due to the new stem and leaf growth. This Type 4 symptom sequence change was associated with high precipitation in the area and is referred to as recovery or temporary recovery if new TCD symptoms develop on this tree in the future. This recovery symptom sequence has not been described in the West. Recovery from TCD was also observed in Richmond in 20113.

208 G. J. Griffin Fig. 2. Type 2 TCD symptom sequence (green foliage wilt/dead clinging leaves) observed in 2012 in the north Richmond, VA TCD area. (Top photograph) Green foliage wilt (right side of photograph) observed on 4 May 2012 on RNT5 black walnut tree with previous severe TCD in Richmond. Overall, about 15% of the foliage present exhibited wilt. (Bottom photograph) Dead, clinging leaves (left side of tree), same tree, 1 month later, on June 4, 2012. The tree was rated as having a 40% live crown on May 4, 30% on June 4 and July 2 and was removed by the owner shortly after in July. No leaflet yellowing was observed during 2012 on this tree. 3.3 Late-season yellow foliage symptom development on black walnut trees in a non-tcd area and in TCD areas Late-season leaflet yellowing and leaflet drop were observed in both non-tcd and TCD areas. This yellowing and leaflet drop usually developed in August (although sometimes earlier in July during dry periods) to various degrees on about 25% of the trees, with more trees later in the season, and was usually scattered throughout the crown of trees and not concentrated in the upper tree crown as with TCD. The percentage of leaflets affected usually increased as the late-season progressed. Fig. 5 shows black walnut tree BT2 on 17 August 2012 in the non-tcd area of Blacksburg with these symptoms, which developed every year. Normal leaf development always occurred during the following spring by May with no evidence of dieback (Table 2). At this site, a few trees on a ridgetop on 9 September 2011 had more leaf drop than trees on the lower slope. Examination and dissection of the uppermost branches of the ridgetop trees with a bucket truck showed no beetle holes and cankers in the bark phloem tissues. In TCD areas, this late-season yellowing condition was

Status of thousand cankers disease 209 Fig. 3. Type 3 TCD symptom sequence (upper tree crown yellowing/leaflet drop) observed in 2012 in the north Richmond, VA TCD area on black walnut tree, RNT6. (Top photograph) Progressive and extensive leaflet yellowing followed by leaflet drop, leaf wilting and branch death in the upper tree crown. This was followed by tree death in a short, 1-year-long sequence. In 2012, the tree had an 80% live crown rating on May 4, June 4 and July 2; a 60% rating on August 1, when the photograph was taken, and a 40% rating on 6 September 2012. On 6 May 2013, the tree was dead. A few dead, clinging leaves were observed on this tree on 6 September 2012. This sequence is similar to, but somewhat faster, than typical symptom sequences described in the western United States. (Bottom photograph) Extensive yellowing of leaflets, leaflet drop and incipient wilt in the upper tree crown on 1 August 2012. The nuts on the branches were not dropped. especially common in Knoxville on elevated sites, but recovery typically occurred the following spring by May. This temporary late-season, scattered leaflet yellowing and leaflet drop has often been confused with TCD by grounds workers. 3.4 Precipitation patterns, soil water potentials and black walnut leaf proline concentrations at the three locations in relation to TCD The amounts of precipitation in the Knoxville, Richmond and Blacksburg areas for 3 years are shown in Table 3. Richmond had less precipitation for all 3 years than Knoxville or Blacksburg from January 1 to the end of August or the growing sea-

210 G. J. Griffin Fig. 4. Type 4 TCD symptom change (recovery) observed from 2010 to 2013 in the Knoxville, TN TCD area on black walnut tree, TLST1, at the TCD-founder site. (Top photograph) This tree had a 60% live crown rating on 15 September 2010 with moderate dieback of branches. (Bottom photograph) New stem growth and an abundance of new healthy foliage on this tree gradually increased in 2011, 2012 and 2013 until the amount of live crown was very high (90%) on 29 August 2013. During this same time, as commonly observed in both Tennessee and Virginia, some TCD-killed branches fell to the ground each year and contributed to the healthy appearance of the tree. This Type 4 symptom sequence has not been previously described. son. Richmond was especially low in precipitation in 2011 and 2012, and we found new TCD progression occurred in 2011 2012 on black walnut in Richmond when precipitation from January 1 to the end of August (Table 3) was low (60 64 cm). Conversely, new TCD symptoms were not observed on any black walnut trees in Richmond or Knoxville when the precipitation was higher (79 130 cm). For annual precipitation, Blacksburg had the lowest amount of precipitation for two of the 3 years. In 2011, hurricane Irene in Richmond on August 29 resulted in raising the amount of precipitation for the year. The third year (2013) was about the same for Blacksburg and Richmond in which high levels of precipitation

Status of thousand cankers disease 211 Fig. 5. Scattered late-season leaflet yellowing and leaflet drop in the lower tree crown of tree BT2 on 17 August 2012 in the non-tcd area of Blacksburg, VA. This yellowing condition was present throughout the tree crown. Note that the rachis of some leaves shows the absence of some leaflets. Normal leaf development occurred during the following spring on this tree by May with no evidence of dieback. This temporary late-season leaflet yellowing and leaflet drop can be confused with TCD by grounds workers. Table 3. Precipitation amounts (cm) in 2011, 2012, and 2013 for two time periods in the TCD areas of Knoxville, TN and Richmond, VA, and in the non-tcd area of Blacksburg, VA from January 1 each year to the date indicated. Knoxville area (TCD) Richmond area (TCD) Blacksburg area (non-tcd) vs. vs. vs. Date Amount Normal Amount Normal Amount Normal Year: 2011 Aug 22 1 79.12 2.92 60.22 12.25 72.35 2.03 Dec 31 2 143.87 +22.30 120.75 +10.00 110.95 +7.15 Year: 2012 Aug 31 91.39 +5.79 64.43 12.10 71.60 2.82 Dec 31 136.24 +14.67 92.76 17.99 92.78 11.02 Year: 2013 Aug 31 130.45 +44.86 98.68 +22.76 100.45 +26.31 Dec 31 176.15 +54.58 136.35 +25.60 131.06 +27.26 1 Hurricane Irene occurred on 29 August 2011 in the Richmond area resulting in a high rainfall value at that date, and necessitated the use of the Aug 22 date in late summer instead of August 31. 2 Normal amounts of yearly precipitation for Knoxville, Richmond and Blacksburg are 121.57 cm, 110.75 cm and 103.80 cm, respectively. were obtained. This was associated with increased black walnut foliage levels in Richmond during the growing season. Knoxville had very high levels of annual precipitation for all 3 years, which was associated with increased growth and recovery of black walnut from TCD. Soil water potentials over the 2 years ranged from field capacity ( 0.1 to 0.3 bars) to 4 bars at Knoxville, from field capacity to a stressful 15 bars at Richmond, and from field capacity to 6 bars at Blacksburg (Table 4). The 15 bar value in Richmond was obtained in August, 2012 at the time of new TCD symptom development in Richmond. Soil water potentials in 2013 in Knoxville and Richmond, associated with recovery of black walnut from TCD, were high and ranged from 0.1 to 3 bars. Proline concentrations in black walnut leaves were variable over the 2 years with no apparent association to soil water potentials (Table 4). However, over the two years, the TCD sites in Knoxville and Richmond had significantly (p < 0.01) higher mean proline concentrations, (9.42 9 10 2 lmol proline per g. leaf tissue) than the non-tcd sites in Blacksburg (5.91 9 10 2 lmol proline per g. leaf tissue), based on a t-test (Ott 1993). The highest proline concentration (20. 8 lmol 9 10 2 per g. leaf tissue) was found in leaf samples from the Knoxville TCD site in 2011 having a black walnut tree with severe TCD and the lowest live crown rating (40%) for all trees evaluated for proline over the 2 years (Table 4).

212 G. J. Griffin Table 4. Stress evaluations for eastern black walnut trees using soil water potentials (SWP) and leaf proline concentrations, determined each month, during 2012 and 2013 from May to August 1, in the TCD areas of Knoxville, TN and Richmond, VA, and in the non-tcd area of Blacksburg, VA. Tree/Soil code Year May June July August SWP/proline SWP/proline SWP/proline SWP/proline Knoxville area KLST6 2012 1/12.6 1.4 3 4/20.8 3.9 1/10.4 0.9 0.3/3.3 0.5 SlL 2 2013 0.3/10.8 2.6 ND 4 ND ND KLST7 2012 1/10.1 1.9 3/7.7 1.4 0.3/14.5 2.6 0.3/11.0 3.4 SICL 2013 0.3/9.5 0.7 0.3/10.3 0.9 3/17.4 0.6 CS 5 /17.0 2.1 Richmond area RCHT1 2012 1/12.0 0.6 2/12.5 2.2 0.3/16.5 7.9 3/16.1 0.6 L 2013 1/8.3 0.4 1/4.8 0.4 0.1/6.4 0.6 2/5.2 0.3 RCST2 2012 1/5.2 0.4 0.3/3.0 0.7 0.3/2.7 0.6 15/5.2 2.1 SL 2013 1/3.5 0.3 1/3.8 0.2 0.1/5.1 0.4 1/8.0 1.4 Blacksburg area BVT2 2012 0.3/4.3 0.2 0.3/3.7 1.3 6/3.2 0.9 3/7.2 0.7 SlL 2013 0.1/7.7 0.2 0.1/15.7 1.6 0.1/9.2 0.6 0.1/5.1 0.2 BNRT1 2012 0.1/3.7 1.0 1/8.6 0.5 3/5.0 0.3 3/3.5 0.3 LS 2013 0.1/3.5 0.2 1/3.1 0.3 0.1/4.7 0.8 2/5.9 0.7 1 The last Richmond assays were done slightly later on 6 September 2013. 2 SlL= silt loam, SlCL = silty clay loam, L=loam, SL = sandy loam, LS = loamy sand. 3 Proline concentration expressed in umol proline 9 10 2 per g leaf sample. SWP (in -bars) in the upper 25 cm of the soil profile (feeder root zone) ranged from 0.1 and 0.3 bars (field capacity or no stress) to 15 bars (very stressful). 4 ND = no determinations due to the accidental removal in 2013 by a local arborist of Knoxville research tree, KLST6, having severe disease (a 40% live crown rating in 2011, 2012 and 2013 but with increased foliage density each year). Test tree KLST7 in Knoxville, and test trees RCHT1 and RCST2 in Richmond had 90% live crown ratings and no change in per cent live crown ratings from 2011 to 2013. 5 This soil sample was contaminated with gravel particles from an adjacent road bank which altered the silty clay loam texture and water properties of the soil. 4 Discussion 4.1 Moderate level and quiescent state of TCD in Knoxville and Richmond, 2011 2013 There has been concern that the eastern population of eastern black walnut trees will be devastated or destroyed by TCD. Our data show that starting in 2011 and during most of this study, the numbers or percentages of eastern black walnut trees with slight, moderate and severe TCD (Table 2) were somewhat similar in Knoxville and Richmond and remained that way for 3 years. Of the 106 black walnut trees studied monthly for most of 3 years, 31 trees had moderate to severe TCD, but only three trees exhibited new TCD symptoms leading to greater TCD severity or dieback. Thus, a relatively constant, moderate level of TCD (mean = 76% live crown) was present in these two areas for 3 years and most trees had a quiescent or dormant TCD condition, which has not previously been reported. Further, in both Knoxville and Richmond, TCD is widespread with the quarantine zones extending over nine counties/cities in Virginia (www.vdacs.virginia.gov/plant&pest/ disease-tcd.shtml) and nine counties in Tennessee (http//tn.gov/agriculture/regulatory/tcd.html), suggesting the disease may have been present in these areas for a long time. However, the centres of these two large quarantined areas (which possibly contain some of the oldest TCD sites in the eastern United States.) had many trees with only slight TCD severity ratings over the 3 years of the study (Table 2). The quiescent state of TCD found in these areas suggests that abiotic and biotic environmental stress factors, such as low soil water potentials, as studied here, or possibly other poor site factors may be required to initiate TCD development on individual trees. One additional environmental stress factor that can affect black walnut and canker disease development is frost injury, either early fall or late spring frosts (Berry 1951; Fowells 1965; Griffin 2000; Sinclair et al. 1987). 4.2 Rate of new TCD development, importance of precipitation and types of TCD symptoms observed The slow rate of new TCD development among the black walnuts in our study contrasts with the rapid rate of disease development by another introduced fungal canker pathogen, Cryphonectria parasitica, on an eastern forest nut-tree species, the American chestnut tree. This disease killed approximately 3.5 billion American chestnut trees in about 40 years (Griffin and Elkins 1986; Anagnostakis 1987). Also, the quiescent condition of TCD found in our study is different from the 2 years from initial symptoms to tree death typically observed in the West (Tisserat et al. 2009). However, severe TCD had developed at some time in the past on some of these trees and TCD could lead to more severe disease, under favourable conditions, in the future, as found for two trees in the north Richmond site in 2012. Of the three trees with new symptoms, one died within 1 year and the other two were removed or cut by land owners, mainly because of the presence of hazardous dead branches in the tree crowns.

Status of thousand cankers disease 213 We found new TCD symptoms developed in 2011 and 2012 on trees in Richmond when precipitation from January 1 to the end of August was low (60 64 cm) and not when the precipitation in Richmond or Knoxville was higher (79 130 cm). This does not mean these precipitation values are benchmarks for TCD development, but they form the basis of an hypothesis (which requires further testing) that low precipitation amounts and low soil water potentials (see 4.4 below) favour TCD development. If confirmed, occurrence of normal or above normal precipitation rates over the growing season in these areas may help curtail TCD development and promote tree growth (see 4.3 below). The precipitation that occurs during the growing season or late spring may be more important than precipitation that occurs in the winter. However, winter and early spring precipitation may be essential for maintaining the water table in soils so that soil water can move up in the soil profile by capillarity to the feeder root zone (Lyon et al. 1952). Also, some deep tree roots may help function in supplying water to the tree. The precipitation values found in this study, when no new TCD developed, are much higher than precipitation values reported for most western state areas, where TCD has been found (Tisserat et al. 2011), as indicated by NOAA precipitation reports for these areas. Two of the three trees with new symptoms exhibited TCD symptoms somewhat similar to TCD symptom development in the western United States. Type 2 disease symptoms, green foliage wilt/dead clinging leaves, is similar to that described in the West where dead, clinging leaves were observed in the last stages of TCD (Tisserat et al. 2009). However, no yellowing of foliage was observed at any time during this Type 2 symptom development. Type 3 symptoms, upper tree crown yellowing/leaflet drop, is similar to typical TCD symptom sequences described in the western United States. However, it was faster, as the tree died in 1 year, than the common 2 years reported for the West (Tisserat et al. 2009). 4.3 Recovery of black walnut from TCD, slowed and quiescent dieback, and juxtaposition of trees with new TCD and no new TCD Other black walnut trees exhibited changes in the tree crowns over time of a different nature. This included recovery from TCD on tree KLST1 in Knoxville (Fig. 4), which was mainly associated with high precipitation in the growing season over the last 2 years (Table 3). This new foliage and stem growth or recovery was also observed on other trees in both Richmond and Knoxville in 2013, when precipitation was high (99 and 130 cm), which was manifested by increased amounts and more dense foliage. The duration of this recovery may depend on whether favourable conditions for TCD occur in the future. The slowed and quiescent dieback symptom sequence occurred on branchlets of a large horizontal branch of the TCD-founder tree in the south Richmond location after severe TCD developed on the tree earlier. The quiescent state developed on the branchlets after high precipitation from hurricane Irene in 2011. The timing of severe TCD development on the TCD-founder tree is not known, but based on comments made to us by the tree owner, it was a relatively rapid development of dieback during the previous 2 years. Interestingly, a black walnut tree, located on the same grounds about 40 m distant from the two trees with severe TCD, had a 90% live crown rating on July 5 and 2 August 2011. However, hurricane Irene on 29 August 2011 blew this large tree to the ground. Examination of branches of this relatively healthy tree shortly after the hurricane revealed there were numerous beetle holes and cankers beneath the beetle holes on branches throughout the canopy of this tree. This spatial juxtaposition of severe TCD trees and trees relatively healthy (90% live crown rating) was encountered commonly at TCD study sites in both Richmond and Knoxville, but an explanation for this is not readily apparent. In general, both the Richmond and Knoxville areas commonly had sites with a mix of trees with severe, moderate and slight TCD symptoms. Some of this may possibly be explained by partial resistance to TCD (Utley et al. 2013), but not for the nearby trees with existing 40% live crown ratings. The north Richmond site, where severe new TCD was found in 2012 on two trees, had five additional black walnut trees nearby. Two of these trees had 40% live crown ratings, one tree had a 60% live crown rating, and two trees had a 90% live crown rating in 2012 but no new TCD symptoms developed on these trees. Further research is needed to explain the different patterns of new TCD or no new TCD at the same site. 4.4 Soil water potentials and proline levels versus TCD The north Richmond site, where new TCD symptoms developed, has the same sandy loam soil texture as the RCT2 site where the stressful soil water potential of 15 bars was obtained in August, 2012 (Table 1; Bennett and McClendonk 1905). Sandy loams have lower available water values, compared to the other heavier Richmond or Knoxville soil textures having more silt and clay content. Sandy soils are more likely to be stressful and develop low soil water potentials during low precipitation periods, as found here and this may have contributed to the fast, 1-year death of the black walnut at this site. In Colorado, Tisserat et al. (2009) indicated trees growing on sites prone to drought declined more rapidly from TCD. Sandy alluvial soils, as studied here in the forest area of Blacksburg, in contrast, have a very high water table and are usually not stressful for soil water availability. During black walnut recovery from TCD in 2013 in both areas, high soil water potentials of 0.1 to 3 bars were recorded (Table 4), which are favourable to black walnut health and vigour. The data indicate that higher proline concentrations were associated with black walnut at TCD sites than at non-tcd sites. Proline is considered a plant stress amino acid, which increases in some but not all plants under abiotic stress and has been associated with protective responses to the stress, but the proline increase in response to stress can be slow (Orcutt and Nilsen 2000). Little has been investigated in regard to biotic stress factors, such a disease, however, and proline concentrations. In a recent study on two plant species, proline content increased three- to sixfold upon induction of biotic stress resulting from infection by the bacterium, Ralstonia solanacearum (Sreedevi et al. 2013). It is possible that the

214 G. J. Griffin proline increases in some black walnut leaf tissues of trees infected by G. morbida may have some type of protective role and contribute to the quiescent state of TCD in most black walnuts studied. Further research in this area is needed. 4.5 Scattered leaflet yellowing and drop not related to TCD The results found here indicate that grounds workers should be cautious and not remove black walnut trees when scattered yellowing of leaflets and leaflet drop occur in the lower, middle and upper tree crown (versus extensive yellowing and extensive leaflet drop in the upper tree crown, typical of TCD; compare Figs 3 5) during the late growing season in August. About 25% of black walnut trees in this study, in both TCD and non-tcd areas, developed general yellowing symptoms and leaflet drop of this type which was temporary. Early leaf drop is common for black walnut trees, compared to other hardwoods, and occurs after shoot growth has stopped in late July or August (Fowells1965). The best time to evaluate these types of symptoms, for decisions about tree removal, is during the following spring in May when normal leaf development is expected. 4.6 Future mid-atlantic climate conditions versus TCD Predictions have been made about the future climate conditions in the United States. For the mid-atlantic region, mathematical and meteorological models predict increased annual and winter precipitation amounts for the future, but the predictions for summer are less certain (Ning et al. 2012). If, in some years, drier conditions occur, cultural control measures, such as mesic (moist) site selection for planting and quantitative irrigation to normal or above normal precipitation levels may have promise for management of TCD. Overall, the findings reported here appear favourable for the survival of this threatened tree species in the eastern United States, especially at sites where soil water potential can be managed or remains high as in alluvial soils. Acknowledgements I thank Diane Reaver for technical assistance, and Scott Schlarbaum, University of Tennessee, and the Tennessee Division of Forestry for help in locating eastern black walnut trees in Knoxville. I thank Norm Dart, VDACS, for help in locating eastern black walnut trees in the Richmond area. I thank Mr. William T. Stubbs, landowner, for delaying cutting of the Virginia TCD-founder tree so that studies on TCD could be continued. Work on this disease was conducted under Permit P526-120406-008, Animal and Plant Health Inspection Service, U.SDA. References Anagnostakis, S., 1987: Chestnut blight: the classical problem of an introduced pathogen. Mycologia 79, 23 37. Bennett, H. H.; McClendonk, W. E., 1905: Soil Survey of Hanover County, Virginia, National Conservation Service Resources. http://www. nrcs.usda.gov/internet/fse_manuscripts/virginia/hanoverva1905/hanoverva1905.pdf. Berry, F. H., 1951: Winter injury to Asiatic chestnut trees in the South during November, 1950. Plant Dis. Rptr. 35, 504 505. Braun, E. L., 1950: Deciduous Forests of Eastern North America. Philadelphia: The Blakiston Co., pp. 596. Carillo, P.; Gibon, Y., 2011: Extraction and determination of proline. Protocols in ecological & environmental plant physiology. Prometheus Wiki. 3 pp. http://prometheuswiki.publish.csiro.au/tikiindex.php?page=extraction+and+determination+of+proline Fowells, H. A., 1965: Silvics of Forest Trees of the United States. Agriculture Handbook No. 271. Washington, DC: Forest Service U. S. Department of Agriculture, pp. 762. Griffin, G. J., 2000: Blight control and restoration of the American chestnut. J. Forest. 98, 22 27. Griffin, G. J.; Elkins, J. R., 1986: Chestnut blight. In: Chestnut Blight, Other Endothia Diseases, and the Genus Endothia. Ed. by Roane, M. K.; Griffin, G. J.; Elkins, J. R., St. Paul, MN: APS Press. The American Phytopathological Society, pp. 1 26. Hansen, M. A.; Bush, E.; Day, E.; Griffin, G.; Dart, N., 2011: Walnut thousand cankers disease alert (http://www.ppws.vt.edu/-clinic/alerts/ 07-22-11_TCD_alert.pdf). Blacksburg, Virginia: Virginia Cooperative Extension. Lyon, T. L.; Buckman, H. O.; Brady, N. C., 1952: The Nature and Properties of Soils. New York: The Macmillan Co., pp. 591. Ning, L.; Mann, E.; Crane, R.; Wagneer, T.; Najjar, R.; Singh, R., 2012. Probabilistic projections of anthropogenic climate change impacts on precipitation for the mid-atlantic region of the United States. American Meteorological Society. 25, 5273 5291. Orcutt, D. M.; Nilsen, E. T., 2000: Physiology of Plants under Stress, New York: John Wiley & Sons, Inc., pp. 683. Ott, R. L., 1993: An Introduction to Statistical Methods and Data Analysis. Belmont, CA: Duxbury Press, pp. 1051. Sinclair, W. A.; Lyon, H. H.; Johnson, W. T., 1987: Diseases of Trees and Shrubs, Ithaca, NY: Cornell University Press, pp. 574. Sreedevi, S.; Remani, K. N.; Benjamin, S., 2013: Biotic stress induced biochemical and isozyme variations in ginger and tomato by Ralstonia solanacearum. Am. J. Plant Sci. 4, 1601 1610. Tisserat, N.; Cranshaw, W.; Leatherman, D.; Utley, C.; Alexamder, K., 2009: Black walnut mortality in Colorado caused by the walnut twig beetle and thousand cankers disease. Online. Plant Health Prog. 1 10. doi: 10.1094/PHP-2009-0811-01-RS. Tisserat, N.; Cranshaw, W.; Putnam, M. L.; Pscheidt, J.; Leslie, C. A.; Murray, M.; Hofman, J.; Barkley, Y.; Alexander, K.; Seybold, S. J., 2011: Thousand cankers disease is widespread in black walnut in the western United States. Online. Plant Health Prog. 1 4. doi: 10.1094/ PHP-2011-0630-01-BR. Utley, C.; Nguyen, T.; Roubtsova, T.; Coggeshall, M.; Ford, T. M.; Gauke, L. J.; Graves, D.; Leslie, C. A.; McKenna, J.; Woeste, K.; Yaghmou, M. A.; Seybold, S. J.; Bostock, R. M.; Tisserat, N., 2013: Susceptibility of walnut and hickory species to Geosmithia morbida. Plant Dis. 97, 601 607.