Mechanistic links between physiology and spectral reflectance enable previsual detection of oak wilt and drought stress.

Tree mortality due to global change-including range expansion of invasive pests and pathogens-is a paramount threat to forest ecosystems. Oak forests are among the most prevalent and valuable ecosystems both ecologically and economically in the United States. There is increasing interest in monitoring oak decline and death due to both drought and the oak wilt pathogen (Bretziella fagacearum). We combined anatomical and ecophysiological measurements with spectroscopy at leaf, canopy, and airborne levels to enable differentiation of oak wilt and drought, and detection prior to visible symptom appearance. We performed an outdoor potted experiment with Quercus rubra saplings subjected to drought stress and/or artificially inoculated with the pathogen. Models developed from spectral reflectance accurately predicted ecophysiological indicators of oak wilt and drought decline in both potted and field experiments with naturally grown saplings. Both oak wilt and drought resulted in blocked water transport through xylem conduits. However, oak wilt impaired conduits in localized regions of the xylem due to formation of tyloses instead of emboli. The localized tylose formation resulted in more variable canopy photosynthesis and water content in diseased trees than drought-stressed ones. Reflectance signatures of plant photosynthesis, water content, and cellular damage detected oak wilt and drought 12 d before visual symptoms appeared. Our results show that leaf spectral reflectance models predict ecophysiological processes relevant to detection and differentiation of disease and drought. Coupling spectral models that detect physiological change with spatial information enhances capacity to differentiate plant stress types such as oak wilt and drought.

Vacuum Steam Treatment of Metrosideros polymorpha Logs for Eradication of Ceratocystis huliohia and C. lukuohia.

A new and devastating disease, rapid ohia death (ROD), in Hawaii led to a state quarantine that regulates interisland transport of ohia wood and plant material to prevent spread of the causal pathogens. Heat treatments of ohia logs in commercial trade were considered for phytosanitary treatment. Vacuum steam (VS) was evaluated for its ability to eradicate the pathogens, Ceratocystis lukuohia and C. huliohia, in main stem logs from ROD-affected forest trees. Replicate loads of three debarked logs (24 to 43 cm in diameter, 1.7 to 2.0 m long) were VS treated at 56°C for 30 min (five loads) or 60°C for 60 min (four loads) at a sapwood depth equal to 70% of log radius. Percentage isolation of Ceratocystis from VS and ambient temperature logs before treatment and summarized by source tree ranged from 12 to 66% and 6 to 31% based on carrot baiting assays of tissue taken from outer and inner sapwood, respectively. No viable Ceratocystis was detected in sapwood locations for the 60°C/60 min schedule or inner locations for the 56°C/30 min schedule after treatment. Only one subsample (0.48%, n = 208) of the latter schedule treatment yielded Ceratocystis. Time needed for treatment ranged from 7.4 to 15 h for the 56°C/30 min schedule and from 8.6 to 19.2 h for the 60°C/60 min schedule. These results demonstrate that VS is an effective and efficient method for treating large-diameter ohia logs that mill owners and regulatory plant pathologists may consider for use in Hawaii.

Pathogenicity, Symptom Development, and Colonization of Metrosideros polymorpha by Ceratocystis lukuohia.

Extensive mortality of Metrosideros polymorpha (`ohi`a) trees has been associated with Ceratocystis spp. on Hawai`i Island and was named rapid `ohi`a death (ROD). Both C. lukuohia and C. huliohia have been associated with ROD, although C. lukuohia appears to be the more important pathogen. Crown observations and dissections of forest trees either wound-inoculated with, or naturally infected by, C. lukuohia were conducted to confirm pathogenicity and document patterns of host colonization. In pathogenicity trials, one of three and two of three trees inoculated with the fungus in February and August, respectively, exhibited crown wilt symptoms at 92 and 69 days after inoculation. Extensive, radial, black staining of the sapwood was found in main stems, while no crown wilt or xylem staining was found in control trees. Xylem staining, necrotic phloem, and fungus presence was noted in six trees inoculated in May to June and harvested 37 to 42 days later, and these observations were compared with those in two naturally infected trees felled in early August. Contiguous xylem staining was found in the main stems and into crowns of all diseased trees, while discontinuous streaks of xylem staining extended into the main forks and side branches. Necrotic phloem associated with xylem staining occurred on the lower stems of inoculated trees. Aside from the necrotic phloem and radial staining of the sapwood, symptom development in `ohi`a infected with C. lukuohia is similar to other systemic wilt diseases on hardwood trees. We propose Ceratocystis wilt of `ohi`a as the official name of the disease.

Vacuum Steam Treatment Eradicates Viable Bretziella fagacearum from Logs Cut from Wilted Quercus rubra.

Methyl bromide (MB) fumigation is required for U.S. Quercus log exports due to concern over unintentional spread of Bretziella fagacearum. MB alternatives are needed due to the chemical's ability to damage the earth's ozone layer. Vacuum steam (VS) is an environmentally friendly method that was evaluated for its ability to eradicate B. fagacearum in logs (24 to 61 cm diameter; 1.7 to 1.9 m long) obtained from Q. rubra that had wilted following natural infection (NI) or artificial inoculation (AI). Five replicate loads of two NI and one AI logs were VS-treated at 56°C for 30 min and 60°C for 60 min (at 5.0 cm sapwood depth). Mean frequencies of pretreatment fungus colonization ranged from 12.5 to 24.4% in NI and 29.4 to 45.6% in AI logs based on isolation from wood chips of inner and outer sapwood, respectively, of two disks per log. Frequencies of pathogen DNA detection were similar to those for isolation. No viable pathogen or its DNA were detected in posttreatment logs. Treatment times ranged from 5 to 9 h for the 56°C/30 min schedule and from 8 to 10 h for the 60°C/60 min schedule. Based on these results, VS is worthy of further development as a MB alternative.

Vacuum Steam Treatment Effectiveness for Eradication of the Thousand Cankers Disease Vector and Pathogen in Logs From Diseased Walnut Trees.

Logs of high-value eastern black walnut (Juglans nigra L.) are commonly exported from the United States for production of veneer and lumber. Veneer logs are not debarked to minimize degradation of wood quality and reduce moisture loss. Thousand cankers disease (TCD) is caused by the walnut twig beetle (Pityophthorus juglandis Blackman) and the fungal pathogen, Geosmithia morbida M. Kolarik, E. Freeland, C. Utley and N. Tisserat sp. nov., which colonize the inner bark of Juglans species. Effective eradication of these organisms by heat or chemical fumigation treatment is required for walnut logs prior to export. Because vacuum steam is an effective and efficient means of heating round wood, its use in eliminating the TCD causal agents was evaluated using Juglans logs (12- to 44-cm small end diameter and 1.7- to 1.9-m length) from TCD-symptomatic trees in Oregon and Washington State. Five replicate trials with three logs per load were conducted in a portable vacuum chamber to test two treatment schedules: 60°C for 60 min and 56°C for 30 min. Complete elimination of P. juglandis and G. morbida was achieved when using a minimum of 56°C at 5-cm targeted depth from bottom of bark furrow into the sapwood and held for 30 min. Treatment cycle time ranged from 298 to 576 min depending on log diameter and initial log temperature. Artificial inoculation of J. nigra trees with G. morbida within the TCD range in Pennsylvania was minimally successful in producing adequately colonized logs for experimental trials.

Propiconazole distribution and effects on Ceratocystis fagacearum survival in roots of treated red oaks.

We investigated the interaction between the oak wilt pathogen (Ceratocystis fagacearum) and propiconazole in lower stems and roots of Quercus rubra to better understand published reports of fungicide failure after 2 years. Propiconazole was infused into mature oaks in July 2004 and roots were inoculated with pathogen endoconidia 1.0 m from injection sites at ±2 weeks of fungicide treatment. Pathogen presence in wood samples was determined by isolation and fungicide concentrations measured using gas chromatography-mass spectrometry. Propiconazole was detected in the roots (≤1.0 m from injection sites) of all treated trees at 2, 12, and 24 months. Propiconazole was detected in all samples (n=68) at 2 and 12 months and in 93% of samples (n=72) at 24 months with concentrations ranging from 815 ppm (12 months in lower stem) to 0.7 ppm (24 months in most distal root segment). Although pathogen isolation incidence was lower in treated than disease control trees at 2 and 12 months, at no time did an infused oak fail to yield the fungus upon isolation. The results document basipetal movement and degradation of propiconazole, as well as the survival of the pathogen, over time in roots and lower stems of infused red oaks.

The origin of Ceratocystis fagacearum, the oak wilt fungus.

The oak wilt pathogen, Ceratocystis fagacearum, may be another example of a damaging, exotic species in forest ecosystems in the United States. Though C. fagacearum has received much research attention, the origin of the fungus is unknown. The pathogen may have been endemic at a low incidence until increased disturbances, changes in land use, and forest management created conditions favorable for disease epidemics. The host genus Quercus contains some relatively resistant species native to the United States, further supporting the hypothesis that the pathogen is native in origin. However, there are also many common, highly susceptible Quercus species--a characteristic typical of introduced pathogens. Most convincingly, studies have shown that the known populations of C. fagacearum have experienced a severe genetic bottleneck that can only be explained by a single introduction. The weight of evidence indicates that C. fagacearum is an introduced pathogen, with possible origins in Central or South America, or Mexico.

Spectral differentiation of oak wilt from foliar fungal disease and drought is correlated with physiological changes.

Hyperspectral reflectance tools have been used to detect multiple pathogens in agricultural settings and single sources of infection or broad declines in forest stands. However, differentiation of any one disease from other sources of tree stress is integral for stand and landscape-level applications in mixed species systems. We tested the ability of spectral models to differentiate oak wilt, a fatal disease in oaks caused by Bretziella fagacearum ``Bretz'', from among other mechanisms of decline. We subjected greenhouse-grown oak seedlings (Quercus ellipsoidalis ``E.J. Hill'' and Quercus macrocarpa ``Michx.'') to chronic drought or inoculation with the oak wilt fungus or bur oak blight fungus (Tubakia iowensis ``T.C. Harr. & D. McNew''). We measured leaf and canopy spectroscopic reflectance (400-2400 nm) and instantaneous photosynthetic and stomatal conductance rates, then used partial least-squares discriminant analysis to predict treatment from hyperspectral data. We detected oak wilt before symptom appearance, and classified the disease with high accuracy in symptomatic leaves. Classification accuracy from spectra increased with declines in photosynthetic function in oak wilt-inoculated plants. Wavelengths diagnostic of oak wilt were only found in non-visible spectral regions and are associated with water status, non-structural carbohydrates and photosynthetic mechanisms. We show that hyperspectral models can differentiate oak wilt from other causes of tree decline and that detection is correlated with biological mechanisms of oak wilt infection and disease progression. We also show that within the canopy, symptom heterogeneity can reduce detection, but that symptomatic leaves and tree canopies are suitable for highly accurate diagnosis. Remote application of hyperspectral tools can be used for specific detection of disease across a multi-species forest stand exhibiting multiple stress symptoms.

Effect of Bark Application With Beauveria bassiana and Permethrin Insecticide on the Walnut Twig Beetle (Coleoptera: Curculionidae) in Black Walnut Bolts.

Formulations of entomopathogenic (insect-killing) fungi represent alternatives to synthetic insecticides in the management of forest and shade tree insects. We evaluated bark spray applications of the entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) strain GHA (BotaniGardES), permethrin insecticide (Astro), and water (control) on colonization of black walnut (Juglans nigra L.) (Fagales: Juglandaceae) bolts by the walnut twig beetle (Pityophthorus juglandis Blackman) (Coleoptera: Curculionidae), vector of the fungus that causes thousand cankers disease. Treated bolts were baited with a P. juglandis aggregation pheromone lure and deployed in infested walnut trees. Bark application of permethrin prevented P. juglandis colonization of the phloem. Although treatment of bolts with the B. bassiana suspension did not reduce P. juglandis colonization or short-term emergence relative to the control treatment, it increased the B. bassiana infection rate from 25 to 62% of emerged adults. Results suggest that commercial applications of B. bassiana strain GHA may help augment natural levels of infection by this entomopathogen in the eastern United States, and support continued exploration of entomopathogens for biological control of the walnut twig beetle.

Three Colopterus Beetle Species Carry the Oak Wilt Fungus to Fresh Wounds on Red Oak in Missouri.

Beetles in the family Nitidulidae can transmit the oak wilt fungus, Ceratocystis fagacearum, to fresh wounds on healthy oak trees, leading to infection and disease development. Historically, nitidulid beetles have not been considered important vectors of the pathogen in Missouri. Studies were conducted in the spring of 2005 and 2006 to determine frequencies of nitidulid beetle species contaminated with C. fagacearum visiting fresh wounds on red oak trees in central Missouri. Colopterus truncatus, C. niger, and C. semitectus were the most abundant species collected from fresh wounds and the only species found to be contaminated with Ceratocystis fagacearum. Of 230 beetles assayed for C. fagacearum, 23 yielded the fungus. Contamination frequencies were higher for beetles collected in April than May; no beetles collected in June were contaminated. We hypothesize that Colopterus truncatus, C. niger, and C. semitectus are principal nitidulid beetle vector species in Missouri during spring. The risk for pathogen transmission by these beetles appears to be greatest in April and least in June.

Seasonal and Regional Distributions, Degree-Day Models, and Phoresy Rates of the Major Sap Beetle (Coleoptera: Nitidulidae) Vectors of the Oak Wilt Fungus, Bretziella fagacearum, in Wisconsin.

Oak wilt is a lethal disease caused by the invasive fungus Bretziella fagacearum, which is transmitted belowground via root grafts and aboveground by sap beetles (Nitidulidae). Attempts to limit spread and impact of B. fagacearum emphasize limiting harvesting and pruning to periods of vector inactivity. However, there is limited information on sap beetle activity periods, responses to temperature, and phoresy frequencies of fungi. We sampled two major vectors in Wisconsin, Colopterus truncatus and Carpophilus sayi, for 2 yr to quantify their seasonal and geographic abundances. Trapping was performed in 12 oak stands, and beetles were assayed for B. fagacearum. C. truncatus was captured from March until November, peaking during April and May. C. sayi was captured from April until November, peaking in May and July. Relative abundances (N = 15,980) were 59.3% C. truncatus and 40.7% C. sayi. C. sayi was more abundant in southern Wisconsin, whereas C. truncatus was more evenly distributed. Both species were present at asymptomatic sites. All sites with oak wilt centers yielded beetles with viable fungal propagules, with the frequency of association ranging from 1 to 50%. Sites asymptomatic for oak wilt contained both beetle species, but no vector-borne viable pathogen. Degree-day models were constructed to improve the generality of these results and estimate cumulative emergences across a latitudinal range over the previous 10-yr means and extremes. Because activity by C. truncatus and C. sayi spans the seasonal activities of oak wilt vectors, these results can help guide oak management practices.

Rapid and PCR-free DNA Detection by Nanoaggregation-Enhanced Chemiluminescence.

The aggregation of gold nanoparticles (AuNPs) is known to induce an enhancement of localized surface plasmon resonance due to the coupling of plasmonic fields of adjacent nanoparticles. Here we show that AuNPs aggregation also causes a significant enhancement of chemiluminescence in the presence of luminophores. The phenomenon is used to introduce a rapid and sensitive DNA detection method that does not require amplification. DNA probes conjugated to AuNPs were used to detect a DNA target sequence specific to the fungus Ceratocystis fagacearum, causal agent of oak wilt. The hybridization of the DNA target with the DNA probes results in instantaneous aggregation of AuNPs into nanoballs, leading to a significant enhancement of luminol chemiluminescence. The enhancement reveals a linear correlation (R2 = 0.98) to the target DNA concentration, with a limit of detection down to 260 fM (260 × 10-15 M), two orders of magnitude higher than the performance obtained with plasmonic colorimetry and absorption spectrometry of single gold nanoparticles. Furthermore, the detection can be performed within 22 min using only a portable luminometer.

Fumigant distribution in forest nursery soils under water seal and plastic film after application of dazomet, metam-sodium and chloropicrin.

Adequate concentration, exposure time and distribution uniformity of activated fumigant gases are prerequisites for successful soil fumigation. Field experiments were conducted to evaluate gas phase distributions of methyl isothiocyanate (MITC) and chloropicrin (CP) in two forest-tree nurseries. Concentrations of MITC and CP in soil air were measured from replicated microplots that received dazomet, metam-sodium and CP. Half of the plots were covered with high-density polyethylene tarp immediately after fumigation; the other half were not covered but received daily sprinkler irrigation for 1 week to create and maintain a water seal. The magnitude of MITC concentrations was similar between nurseries for metam-sodium in both tarp and water seal treatments and for dazomet in the tarp treatment. Consistently greater MITC and CP concentrations were found in the upper 30 cm of soil in the tarped plots compared with the water-sealed plots. Despite potential environmental and economic benefits with the water seal method, tarp covers were more reliable for achieving and maintaining higher MITC and CP concentrations and less prone to variations due to irrigation/rain, soil bulk density and other environmental conditions.

Seasonal Dispersal of the Oak Wilt Fungus by Colopterus truncatus and Carpophilus sayi in Minnesota.

Sap beetles (Nitidulidae) are considered important overland vectors of the oak wilt pathogen, Ceratocystis fagacearum, in the north central United States. Colopterus truncatus and Carpophilus sayi are thought to be the principal sap beetle vectors in Minnesota. Field studies using windoriented funnel traps baited with aggregation pheromones of the insects were conducted during 2 years in east central Minnesota. The studies compared temporal flight dynamics of Colopterus truncatus and Carpophilus sayi from April through October, measured the proportion of dispersing adults of each species carrying viable pathogen propagules, and estimated the populations of contaminated dispersing beetles in oak (Quercus spp.) stands with and without oak wilt. Abundance of Colopterus truncatus peaked in either April or May, but the pathogen was most frequently isolated (20 to 45%) from beetles obtained from oak wilt sites from July through September. However, the highest contaminated insect population (CIP) generally occurred during April and May. Abundance of Carpophilus sayi peaked in October of both years, but the pathogen was most commonly isolated from beetles collected during May and June. The CIP was highest in June. The infective insect potential, as indexed by CIP, was greatest for both species in the spring and was greater for Colopterus truncatus than for Carpophilus sayi.

Colonization of Artificially Stressed Black Walnut Trees by Ambrosia Beetle, Bark Beetle, and Other Weevil Species (Coleoptera: Curculionidae) in Indiana and Missouri.

Thousand cankers disease (TCD) is a new disease of black walnut (Juglans nigra L.) in the eastern United States. The disease is caused by the interaction of the aggressive bark beetle Pityophthorus juglandis Blackman and the canker-forming fungus, Geosmithia morbida M. Kolarik, E. Freeland, C. Utley & Tisserat, carried by the beetle. Other insects also colonize TCD-symptomatic trees and may also carry pathogens. A trap tree survey was conducted in Indiana and Missouri to characterize the assemblage of ambrosia beetles, bark beetles, and other weevils attracted to the main stems and crowns of stressed black walnut. More than 100 trees were girdled and treated with glyphosate (Riverdale Razor Pro, Burr Ridge, Illinois) at 27 locations. Nearly 17,000 insects were collected from logs harvested from girdled walnut trees. These insects represented 15 ambrosia beetle, four bark beetle, and seven other weevil species. The most abundant species included Xyleborinus saxeseni Ratzburg, Xylosandrus crassiusculus Motschulsky, Xylosandrus germanus Blandford, Xyleborus affinis Eichhoff, and Stenomimus pallidus Boheman. These species differed in their association with the stems or crowns of stressed trees. Multiple species of insects were collected from individual trees and likely colonized tissues near each other. At least three of the abundant species found (S. pallidus, X. crassiusculus, and X. germanus) are known to carry propagules of canker-causing fungi of black walnut. In summary, a large number of ambrosia beetles, bark beetles, and other weevils are attracted to stressed walnut trees in Indiana and Missouri. Several of these species have the potential to introduce walnut canker pathogens during colonization.

Colonization of Oak Wilt Fungal Mats by Ophiostoma piceae During Spring in Minnesota.

The colonization of Ceratocystis fagacearum fungal mats of different ages by Ophiostoma piceae on Quercus spp. was determined in three east-central Minnesota locations during the spring of 1995. The extent of the mat area colonized by O. piceae generally increased with mat age. Subsamples per mat yielding the fungus for the three locations averaged 11 to 27% for immature mats, 65 to 72% for mature mats, and 66 to 96% for aging and declining mats. On a mat-incidence basis, frequencies of O. piceae isolation from at least one subsample of an immature mat ranged from 30 to 53% compared with more than 90% for all other mat ages in the three locations. The average number of colony-forming units of O. piceae per square centimeter ranged from 1 × 103 to 1.2 × 106 and increased with mat age. The extent of C. fagacearum recovery from each mat was lower for aging plus declining mats (P < 0.03) compared with other ages in two locations. On a mat-incidence basis, frequency of C. fagacearum recovery from at least one subsample of a mat averaged 99% for all ages of mats in all locations. The interaction between O. piceae and C. fagacearum on mats, nitidulids, and the oak wound surface is discussed.

Acquisition of Ophiostoma quercus and Ceratocystis fagacearum by Nitidulids from O. quercus-Colonized Oak Wilt Mats.

Field experiments were conducted to determine whether the frequency of Ceratocystis fagacearum and Ophiostoma quercus propagule acquisition by nitidulids visiting oak wilt fungal mats is affected by the presence of O. quercus on the mats. Augmentation sprays with O. quercus were used to achieve different levels of mat colonization by that fungus. The extent of colonization by O. quercus 7 to 14 days after spraying was greatest for postmature mats with open cracks (>0.5 cm) observed on the spray date (P< 0.02). Specifically, all six subsamples assayed per mat for 16 of 18 open, O. quercus-sprayed, postmature mats yielded O. quercus. The frequency of nitidulids with O. quercus was also highest (83%) for those collected from O. quercus-treated, postmature mats that were open on the spray date. The frequency of nitidulids with viable C. fagacearum (88%), however, was also greatest for beetles from the postmature mats. Thus, O. quercus colonization did not affect vector acquisition of C. fagacearum from the mats. In addition, O. quercus vectored by nitidulids probably does not result in natural biological control of overland transmission of C. fagacearum at fresh wound surfaces on healthy oaks, as previously suggested.

Semiochemical-mediated flight responses of sap beetle vectors of oak wilt, Ceratocystis fagacearum.

The sap beetle, Colopterus truncatus (Coleoptera: Nitidulidae), is one of the primary vectors of the oak wilt pathogen, Ceratocystis fagacearum, in the north-central United States. Field behavioral assays utilizing various release rates and blends of three methyl-branched hydrocarbon aggregation pheromone components showed that flight responses of this beetle were similar in Illinois and Minnesota populations. In both locations, both sexes of the beetle responded synergistically to a combination of the three-component pheromone and fermenting whole-wheat bread dough. Further, Colopterus truncatus preferred a high release rate over a low release rate of the three-component blend. In both locations, the response of C. truncatus to a simplified version of the pheromone consisting of (2E,4E,6E)-3,5-dimethyl-2,4,6-octatriene (1) and (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene (3) was not significantly different from the response to the three-component blend. An experiment in Illinois with all possible combinations of the components demonstrated that the decatetraene (3) was the crucial component in the blend; of all treatments, the maximal response was elicited by 3 + dough. Chipped bark, phloem, and xylem from northern pin oak, Quercus ellipsoidalis, was not attractive to C. truncatus in Minnesota. During a weekly survey over two seasons in Minnesota, C. truncatus flew in response to the three-component pheromone between early April and early July, with the maximum responses coming on May 4, 2000 and April 20, 2001. During both years, more than 98% of the beetles were trapped between April 14 and June 1. During the same survey, Glischrochilus spp. (Nitiduildae) flew during longer periods of the summer, particularly in 2001. The sex ratio of C. truncatus responding during all experiments was female-biased (1.8:1, female-male), which is characteristic of other male-produced coleopteran aggregation pheromones. Other sap beetles that play a minor role in the pathobiology of C. fagacearum also responded in experiments conducted in Minnesota. Carpophilus brachypterus Say was cross-attracted to the two- and three-component blends of the C. truncatus pheromone and dough, whereas two Glischrochilus spp. were attracted to all treatments that contained dough.