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Genetic resistance is the most recommended measure to control verticillium wilt in olive (VWO), a vascular disease caused by the soil-borne fungus Verticillium dahliae, which has promoted the development of olive breeding programs aimed at obtaining new resistant and highly yielding cultivars in recent years. Screening has been commonly performed under controlled conditions in grow chamber after artificial inoculation during the early stage of breeding programs, but additional evaluation is necessary to confirm previous results as well as to test for additional agronomic traits. During this study, 20 breeding selections initially classified as resistant to the disease have been re-evaluated in artificially infested soils under natural environmental conditions. The maximum disease incidence (52.6%) was reached at 26 months after planting, and the disease intensity index reached the maximum value of 38.5% at 29 months after planting. Nine breeding selections consistently confirmed the previous results regarding resistance to V. dahliae infection; however, contradictory results, compared with those of previous evaluations under controlled conditions in grow chambers, were obtained for the rest of selections tested, thereby underlining the need for long-term experimentation under natural environmental conditions. Additional positive agronomic traits, such as early bearing, were also observed for some of the resistant selections, but plant vigor varied. Some seem highly promising for release as new cultivars when characterization of other important agronomic traits is completed in the future.

Soilborne diseases caused by pathogens such as Phytophthora, Rhizoctonia, Fusarium, Verticillium, and Pythium species are the most important diseases of woody ornamentals. Ginkgo (Ginkgo biloba) and red maple (Acer rubrum ‘October Glory’) plants grown in containers and fields in Tennessee showed root and crown rot symptoms with dark brown to black lesions in 2017 and 2018. The objective of this research was to isolate and identify pathogens affecting ginkgo and red maple plants in Tennessee nurseries and to develop fungicide/biofungicide management recommendations for nursery producers. Isolations were made from the infected roots. Several Phytophthora-like colonies with spherical zoospores, filamentous to globose oogoni, and whitish mycelium were isolated on V8-PARPH medium. To confirm identity, total genomic DNA was extracted, followed by sequence analysis of the internal transcribed spacer regions, large subunit of nuclear rRNA, and cytochrome c oxidase subunits I and II of mitochondrial DNA. Based on morphological and molecular analysis, Phytopythium vexans was described as a causal agent of crown and root rot from the infected ginkgo and red maple plants. To complete Koch’s postulates, a pathogenicity test was performed by drenching 100 ml of V8 agar medium slurry of Phytopythium vexans inoculum on 1-year-old potted ginkgo plant root systems as well as red maple October Glory. Necrotic lesion development was observed in the root system 45 days after inoculation and Phytopythium vexans was reisolated from the roots of both ginkgo and red maple. All control ginkgo and red maple plants remained disease free and no pathogen was reisolated. In addition, the efficacy of fungicides, biofungicides, fertilizer, and host plant defense inducers (traditionally recommended for management of oomycete diseases) for control of Phytopythium crown and root rot was evaluated on ginkgo and red maple October Glory seedlings in greenhouse and field trials. Fungicides such as Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX were effective in both greenhouse and field trials, and the biofungicide Stargus reduced disease severity caused by pathogen Phytopythium vexans on ginkgo and red maple plants in greenhouse trials. These results will help nursery producers make proper management decisions for newly reported Phytopythium crown and root rot disease of ginkgo and red maple plants.

Verticillium wilt, caused by Verticillium dahliae, is one of the major diseases of eggplants. Nanoparticles (NPs) of CuO, Mn2O3, and ZnO were sprayed alone onto leaves of young eggplants and in different combinations and rates, and then seedlings were transplanted into soil infested with V. dahliae in the greenhouse and field between 2015 and 2018. All combinations of NPs were consistently less effective than CuO NPs applied alone at 500 µg/ml at increasing disease suppression, biomass, and fruit yield. CuO NPs were associated with an increase in fruit yield (17 and 33% increase) and disease suppression (28 and 22% reduction) in 2016 and 2017, respectively, when compared with untreated controls. However, this effect was negated in the greenhouse and field experiments when CuO NPs were combined with Mn2O3. Combining NPs of CuO with ZnO resulted in variable effects; amendments increased growth and suppressed disease in greenhouse experiments, but results were mixed in the field. Leaf tissue analyses from the greenhouse experiments showed that Cu concentration in leaves was reduced when CuO NPs were combined with other NPs, even when application rates were the same amount. A simple competition for entry sites may explain why combinations of CuO NPs and Mn2O3 NPs reduced efficacy but does not explain the lack of inhibition between Cu and Zn. NPs of CuO performed better than their larger bulk equivalent, and studies on application rate found 500 µg/ml was optimal. No phytotoxicity, as determined, by leaf burning, necrotic spots, or dead apical buds was noted even at the highest combined rates of 1,500 µg/ml.

A highly pathogenic fungus characterized as

The utility of traditional methods for detecting seed-borne fungi is limited by the fact some fungi are unculturable or difficult to isolate. The seed-borne pathogens affecting Panax ginseng cultivation have not been fully characterized. Seed-borne fungi can be identified based on the high-throughput sequencing of internal transcribed spacer (ITS) amplicons. A hierarchical clustering tree diagram analysis based on operational taxonomic units revealed a relationship between the seed-borne fungi and the region from which the seeds were collected. This study analyzed the fungal diversity on 30 ginseng seed samples from the main ginseng-producing areas of China. The 50 most abundant genera were identified including those responsible for ginseng diseases, Fusarium, Alternaria, Nectria, Coniothyrium, Verticillium, Phoma, and Rhizoctonia. Fusarium species, which are the primary causes of root rot, were detected in all seed samples. The results of a phylogenetic analysis indicated that the seed-borne fungal species originating from the same region were closely related. Fungi on ginseng seeds from eight different regions were divided into eight clades, suggesting they were correlated with the local storage medium. A total of 518 Fusarium isolates were obtained and 10 species identified, all of which can be detrimental to ginseng production. Pathogenicity tests proved that seed-borne Fusarium species can infect ginseng seedlings and 2-year-old ginseng root, with potentially adverse effects on ginseng yield and quality.

In August 2019, Verticillium wilt was observed in commercial alfalfa fields in Jinta County, Jiuquan, located to the west of Gansu, China, where Verticillium wilt of alfalfa was first observed in this region. This study was conducted to evaluate the effect of alfalfa cultivars (Galaxie Max, Liangmu No. 2, and Danon VNS, planted in 2017) and ages (cultivar Adrenalin, planted in 2014, 2015, and 2016) on the occurrence of Verticillium wilt caused by

Verticillium longisporum is a soilborne vascular fungal pathogen that has spread throughout the European oilseed rape cultivation area since the 1980s and was detected in canola fields in Canada in 2014. In a series of greenhouse and field inoculation experiments using V. longisporum-resistant and susceptible cultivars of winter and spring types of oilseed rape, the present study investigated the potential of V. longisporum dissemination by seeds of Brassica napus. Greenhouse inoculation studies with a DsRed-labeled isolate of V. longisporum confirmed the systemic growth of the pathogen from roots to seeds. Further monitoring of plant colonization in the greenhouse with a species-specific real-time polymerase chain reaction assay verified the pathogen growth from roots to stem bases, pods, and seeds in root-inoculated plants. The frequency of recovery of viable colonies of V. longisporum from seeds harvested from greenhouse-grown inoculated plants ranged from 0.08 to 13.3%. The frequency of seed transmission in the greenhouse differed in oilseed rape cultivars varying in susceptibility to V. longisporum. Subsequent studies on transmission of the disease into the offspring revealed that only 1.7 to 2.3% of plants showed disease symptoms as confirmed by the formation of microsclerotia in the stems. Results from field-grown plants differed from the greenhouse studies. The degree of seed transmission in the field was dependent on the crop type. Although only low concentrations of DNA of V. longisporum were detectable in seeds harvested from severely infected winter oilseed rape, significantly greater concentrations of fungal DNA were found in seeds of spring-type oilseed rape, at similar soil conditions and inoculum densities. Correspondingly, plating seeds that were harvested from infected plants on agar yielded viable V. longisporum colonies only from seeds of the spring-type but not of the winter-type plants. Lack of seed infection in the winter-type crop was confirmed in two seasons. Equally, none of the offspring grown from seeds from severely diseased winter oilseed rape plants developed symptoms of Verticillium stem striping. The results suggest that the rate of seed transmission of V. longisporum depends on the degree of plant colonization, which is significantly faster under greenhouse than field conditions and in a spring-sown crop compared with an autumn-sown oilseed rape crop. According to our studies, disease transmission by seeds from European winter oilseed rape production cannot be confirmed.

Verticillium wilt caused by Verticillium alfalfae results in severe production losses in alfalfa crops and is a Class A quarantined disease in China. During 2015 to 2017, 365 alfalfa fields from 21 locations in six provinces were surveyed, and 45 fields from three closely located sites in Gansu, China were found to have alfalfa plants with symptoms typical of Verticillium wilt, with disease incidence of 12.6 to 53.6%. Isolates were identified to species using morphological characteristics and a maximum likelihood phylogeny of the concatenated partial sequences of actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase, and tryptophan synthase gene regions of Verticillium isolates. Isolation incidence was 93.9% from roots, 71.7% from stems, 66.1% from petioles, and 32.2% from leaves of field-infected plants, indicative of systemic disease and sporadic distribution of this pathogen. In greenhouse tests, the pathogen infected seedlings and colonized vascular tissues when inoculated on seeds, on root tips, in soil, or in injured, but not uninjured, aerial tissues, causing systemic symptoms like those in the field and significant losses. Pathogenicity testing also revealed that five locally grown perennial legumes (stylo, milkvetch, sainfoin, white clover, and red clover) could host V. alfalfae, with a high virulence to milkvetch, sainfoin, and stylo. This study confirmed that V. alfalfae has become established in some regions of Gansu, China and that is a risk to the alfalfa industry in China.

Verticillium wilt caused by Verticillium spp., also called potato early dying disease, is one of the most serious soilborne diseases affecting potato production in China. The disease has been expanding into most potato production areas over the past few years. Information on host resistance against Verticillium wilt among the potato cultivars in China is scarce, but it is critical for sustainable management of the disease. This study, therefore, evaluated 30 commercially popular potato cultivars against Verticillium dahliae strain Vdp83 and Verticillium nonalfalfae strain Vnp24, two well-characterized strains causing Verticillium wilt of potato in China. Both strains were isolated from diseased potato plants, and they were previously proven to be highly virulent. Ten plants of each cultivar were inoculated with the V. dahliae strain and incubated on greenhouse benches. Symptoms were rated at weekly intervals, and the relative area under the disease progress curve was calculated. The experiment was repeated once, and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals. Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V. nonalfalfae strain. Cultivar responses to V. nonalfalfae were like those exhibited against V. dahliae, except for one cultivar. This study showed that resistance among potato cultivars exists in China against Verticillium spp. and that the resistance to V. dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions. Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study.

Cotton production in Xinjiang, the largest cotton-producing area in China, has an increasingly serious disease threat from

Verticillium wilt (VW) of alfalfa is a devastating disease that causes forage yield reductions of up to 50% in the northern United States and Canada. The most effective method for controlling the disease is through the development and use of resistant varieties. To identify quantitative trait loci (QTL) for VW resistance in alfalfa, we used a full-sib population segregating for VW resistance. High-density linkage maps for both resistant and susceptible parents were constructed using single-dose alleles of single-nucleotide polymorphism markers generated by genotyping-by-sequencing. Five QTL associated with VW resistance were identified and they were in four linkage groups (4D, 6B, 6D, and 8C). Of those, three QTL (

Verticillium wilt, caused by Verticillium dahliae, is a disease of dicotyledonous crops such as potato and has a wide host range and persistent, long-term survival structures called microsclerotia that can persist in soil for up to 14 years. Some V. dahliae isolates are particularly aggressive on a specific plant host while retaining the ability to infect a wide range of other hosts. Weeds can serve as hosts for V. dahliae but whether they serve as sources of inoculum for aggressive isolates of V. dahliae to crop hosts is unknown. The goal of this research was to quantify V. dahliae microsclerotia obtained from 16 weeds which were grown in the greenhouse. Potting medium was infested with one of eight V. dahliae isolates from potato, mint, sugar beet, sunflower, tomato, and watermelon. The isolates from mint and potato were aggressive on the host from which they were originally isolated. All 16 weeds were infected by at least one V. dahliae isolate, although the number of microsclerotia produced from some infections was relatively low (≤5 microsclerotia/g of dry plant). Black nightshade yielded greater numbers of microsclerotia of the V. dahliae potato isolate than any other isolate in three of four trials in the greenhouse (second trial false discovery rate, adjusted P ≤ 0.0158; third trial, P ≤ 0.0264; and fourth trial, P ≤ 0.0193). Litchi tomato yielded greater numbers of microsclerotia of the V. dahliae potato isolate than any other isolate in one of four trials (first trial, P ≤ 0.0149). A V. dahliae isolate from tomato yielded greater numbers of microsclerotia in large crabgrass and wild oat in a second trial (P ≤ 0.0158). Weeds, depending on the species, grown during and between potato crop rotations may increase the number of microsclerotia of the potato-aggressive isolates of V. dahliae.

Oilseed rape, an important source of vegetable plant oil, is threatened by

Verticillium wilt caused by

Verticillium wilt caused by

Verticillium wilt (Verticillium albo-atrum) is an important disease affecting potato tuber yield and quality. In North America the major commercial cultivars are susceptible and management strategies for control of the pathogen rely mainly on soil fumigation and crop rotation. In this study 398 genotypes from accessions of Solanum berthaultii, S. chacoense, and S. tarijense were screened for resistance to Verticillium albo-atrum. Resistant genotypes were identified in all but two accessions; however, results indicate that tolerance is more common than resistance. We identified two genotypes in S. chacoense (PI 472819) that had low stem-colonization levels and also did not develop wilt symptoms when inoculated with V. albo-atrum. These genotypes and a susceptible genotype from PI 472810 (S. chacoense) were studied to determine genetic inheritance. Segregation ratios in F1, F2, and backcross populations indicated that resistance in one of the resistant genotypes (18-21R) was controlled by a single dominant gene. Transfer of the Vc gene to tetraploid germ plasm could provide effective and economical control of Verticillium wilt.

Solanum cardiophyllum Lindl and Solanum ehrenbergii (Bitt) Rydb are wild edible potato plants found throughout central Mexico (2). These plants are not cultivated, but farmers collect tubers for their own consumption and to sell at local markets (2). Wilted plants were observed in experimental plots of these wild potatoes established near Chapingo, Mexico, during spring 1983. Initial symptoms included wilting and dark yellowing of lower leaves. As the disease advanced, all of the foliage became chlorotic and the plants wilted and eventually died. Disease incidence was 13.4% for S. ehrenbergii and 0.2% for S. cardiophyllum. Verticillium dahliae Kleb. was consistently isolated from the roots and lower stems of diseased plants of both Solanum species. The isolating procedure consisted of thoroughly rinsing roots and lower stems with tap water and cutting roots and stems into 3- to 6-cm sections that were placed in 10% bleach for 3 to 5 min. Bleach excess was removed with sterile paper, and the tissue sections were cut into smaller pieces (0.5 cm) and placed on potato dextrose agar (PDA) plates. Cultures of Verticillium produced numerous dark microsclerotia of various shapes and sizes (0.05 to 0.1 mm); erect, slender, hyaline, and branched conidiophores; and elliptical and hyaline, single-celled conidia characteristic of V. dahliae (1). Pathogenicity studies were conducted in a greenhouse on 2-month-old S. cardiophyllum and S. ehrenbergii plants grown from tubers. Inoculum was obtained from colonies growing on PDA for 10 days producing abundant conidia. Conidial suspensions were obtained by flooding the plate cultures with sterile distilled water, filtering the suspension with two layers of cheesecloth, and adjusting the inoculum to 1.0 × 106 conidia/ml (3). Ten ml of the conidial suspension were applied to each of four holes 5 cm deep and 3 to 5 cm next to the crown of each plant. Symptoms similar to those observed on field-grown plants were observed 15 days after inoculation, and V. dahliae was re-isolated from lower stems and roots. All inoculated plants were dead 4 weeks after inoculation. Water-inoculated plants remained healthy throughout the experiments. This is the first report of V. dahliae on S. cardiophyllum and S. ehrenbergii. References: (1) G. R. Dixon. Vegetable Crop Diseases. Avi Publishing, Westport, Connecticut. 1981. (2) J. Galindo. Naturaleza 13:175, 1982. (3) H. A. Melouk and C. E. Horner. Phytopathology 65:767, 1975.

Isolates of Verticillium dahliae from 15 different hosts and V. albo-atrum from alfalfa were tested for their ability to cause wilt on broccoli using a root-dip inoculation method. None of the isolates caused vascular discoloration in broccoli except those from cabbage and cauliflower that were weakly pathogenic. Broccoli cultivars Baccus, Greenbelt, Parasol, Patriot, and Symphony showed resistance to Verticillium infection. Re-isolated strains from fresh samples of internally discolored broccoli and cauliflower root tissues were unable to cause disease symptoms on re-inoculation of broccoli, but caused severe disease on cauliflower. Inoculation of 5-, 7-, 9-, or 11-week-old plants did not alter the resistance in broccoli or the susceptibility in cauliflower against Verticillium. Immunity of broccoli against infection by isolates of V. dahliae from non-crucifer hosts, its resistance against crucifer isolates, and as previously described, attrition of V. dahliae microsclerotia in soil by broccoli residue, coupled with its importance as a commercial vegetable, make broccoli an attractive rotation crop for the management of Verticillium wilt in many cropping systems.

Two isolates of Verticillium dahliae in vegetative compatibility group (VCG) 2B and one isolate in VCG 4A were tested for aggressiveness on peppermint, Scotch spearmint, and native spearmint alone and in combination with the lesion nematode Pratylenchus penetrans. The VCG 2B isolates were more aggressive than the VCG 4A isolate on all three mint species. Severity of wilt was greatest on peppermint and scotch spearmint with the VCG 2B isolates, and CFU of V. dahliae recovered from infected stems were greater in Scotch spearmint when inoculated with V. dahliae and P. penetrans than when inoculated with VCG 2B alone. Wilt severities and CFU in peppermint and Scotch spearmint were not increased when inoculated with VCG 4A and P. penetrans compared with VCG 4A alone. These data indicate that V. dahliae isolates from VCG 2B but not VCG 4A interact synergistically with P. penetrans on Verticillium isolate-susceptible mints. The resistance to Verticillium isolates in native spearmint was not affected by the presence of lesion nematodes.

Verticillium dahliae is widely distributed in potato and olive fields in Lebanon, causing serious economic losses. However, little is known about the inoculum source, population structure, and genetic diversity of the pathogen or the mechanisms of dissemination within Lebanon. To understand the population structure, a total of 203 isolates sampled from olive (n = 78) and potato (n = 125) were characterized for species, mating type, and race, and the genetic relationships were delineated using 13 microsatellite markers. All isolates except one from potato were V. dahliae, with 55.1 and 12.1% race 1, and 43.6 and 83.1% race 2 in olive and potato, respectively. The genetic structure of the studied population was best described by two large and two small clusters. Membership in the two large clusters was determined by the presence or absence of the effector gene Ave1. Furthermore, genetic structure was moderately associated with the host of origin but was weakly associated with the geographic origin. All but four isolates represented by three multilocus haploid genotypes were MAT1-2. This study identified a clear lack of gene flow between virulence genotypes of V. dahliae despite the proximity of these cropping systems and the wide distribution of genetic diversity among hosts and geographic regions in Lebanon.

There are no previous reports of Verticillium wilt in fresh and processing spinach (Spinacia oleracea) crops in the United States. In 2002, a hybrid spinach seed crop in the Pacific Northwest developed late-season wilt symptoms. Assays of the harvested seed and stock seed of the male and female parents revealed 59.5, 44.0, and 1.5%, respectively, were infected with Verticillium dahliae. Assays of 13 stock or commercial seed lots grown in 2002 and 62 commercial lots harvested in 2003 in Denmark, Holland, New Zealand, and the United States revealed the prevalence of Verticillium spp. in commercial spinach seed. Sixty-eight lots (89%) were infected with Verticillium spp. at incidences ranging from 0.3 to 84.8%. Five spinach seed isolates of V. dahliae were pathogenic on each of three spinach cultivars by root-dip inoculation. V. dahliae was detected on 26.4% of the seed from 7 of 11 inoculated plants but on none of the seed from 6 control plants, demonstrating systemic movement of V. dahliae. Seed-to-seed transmission was also demonstrated by planting naturally infected seed lots. This is the first report of Verticillium wilt of spinach in the primary region of spinach seed production in the United States.

Eggplant cultivars grafted on rootstocks resistant to root-knot nematodes (Meloidogyne spp.) are increasingly grown in Italy to reduce nematode infection. During the winter of 2003-2004, eggplants (cv Black Bell and Mirabell) grafted on the nematode-resistant rootstock Solanum torvum were observed with symptoms of a wilt disease in several greenhouses in Sicily (southern Italy). The vascular tissue in stems of affected plants appeared brown. These plants were stunted and developed yellow leaves with brown or black streaks in the vascular tissue. The wilt appeared in several greenhouses at a very low incidence (0.01 to 0.05%). Later, during the fall of 2004, disease incidence was approximately ten times greater in the same greenhouses on new crops. Verticillium dahliae was consistently and readily isolated from symptomatic vascular tissue of the rootstock (S. torvum) and the scion (cv Black bell) when cultured on potato dextrose agar (PDA) (1). Healthy, 50-day-old plants of S. torvum and eggplant (cv. Black Bell) were separately inoculated by root dip with a conidial suspension (1 × 107 CFU/ml) of two isolates of V. dahliae obtained from the rootstock and the scion of the infected grafted plants and with a known pathogenic isolate of V. dahliae from nongrafted eggplant. Noninoculated S. torvum and eggplant served as control treatments. Plants (30 per treatment) were grown in a glasshouse at temperatures ranging between 12 and 41°C (weekly average 15 to 36°C) and relative humidity ranging between 36 and 99% (weekly average 54 to 95%). The first wilt symptoms and vascular discoloration in the roots, crowns, and veins developed 26 and 21 days after inoculation on S. torvum and eggplant, respectively. Seventy-two days after inoculation, 20, 26, and 27% of S. torvum plants and 97, 100, and 87% of the eggplants showed symptoms caused by V. dahliae isolates obtained from the scion of diseased grafted plants, the rootstock of diseased grafted plants, and nongrafted eggplants, respectively. Noninoculated plants remained healthy. To our knowledge, this is the first report in Italy of Verticillium wilt on eggplant grafted on S. torvum rootstocks under commercial conditions. Use of eggplant grafted on the nematode-resistant rootstock of S. torvum presents an interesting opportunity to control the root-knot nematode but has to be carefully considered when dealing with soils severely infested by V. dahliae. Reference: (1) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

Verticillium isolates collected from lettuce and artichoke were characterized for morphology, growth and pathogenicity. Several isolates were identified as Verticillium tricorpus based on morphological and cultural characteristics, including the production of dark resting mycelia, chlamydospores, microsclerotia, and yellow to orange pigmentation in culture. Compared with isolates of V. dahliae, these isolates also produced microsclerotia and conidia that were significantly larger and exhibited a distinct growth pattern at varying temperatures. Using database sequence information, primers were developed from the internal transcribed spacer region to produce a diagnostic 337-bp product specific to V. tricorpus and used to confirm the identification of isolates. Pathogenicity tests indicated that isolates of V. tricorpus were weak pathogens, causing a median disease severity (DS) of 3.5 on lettuce and 5.0 on artichoke. Although lettuce and artichoke inoculated with isolates of V. tricorpus exhibited reduced height and fresh foliar and root weight, the reductions were not statistically significant, unlike in plants inoculated with isolates of V. dahliae. Lettuce co-inoculated with isolates of V. tricorpus and V. dahliae exhibited reduced symptoms of Verticillium wilt and improved growth relative to those inoculated with V. dahliae alone. The early introduction of V. tricorpus in soil-drench inoculations appeared to provide better relief from subsequent V. dahliae inoculation than when the two species were co-inoculated simultaneously using the root-dip method, suggesting competitive exclusion as a plausible mechanism. A spore-polymerase chain reaction assay developed using cultured spores directly as template and primers specific to V. tricorpus confirmed the presence of V. tricorpus on inoculated roots. This work demonstrates the potential use of V. tricorpus to directly reduce the effect of V. dahliae on lettuce and artichoke and, to our knowledge, is the first reported characterization of V. tricorpus isolates collected from lettuce and artichoke.

Verticillium dahliae (Kleb.) is known worldwide as a destructive soilborne pathogen with a wide host range (2). Reports of V. dahliae attacking cucurbits are generally limited to ‘Casaba’ and ‘Persian’ type melons. During August and September of 2004 to 2006, fields of seedless watermelon (Citrullus lanatus [Thunb.] Matsum. & Nak.) and pollinators in Yoakum County, Texas, exhibited severe symptoms of vine decline. There was no apparent difference between diploid and triploid watermelon cultivars. Night-time temperatures during July, August, and September averaged 20°C or less. Losses were estimated in excess of one-half million dollars. Symptoms consisted of leaf yellowing, wilting, and gradual death of the leaves, but stems generally remained green. The xylem exhibited a uniform tan-to-light brown discoloration that often extended throughout the vine. Dead plants had numerous microsclerotia embedded throughout the root and crown. Crown and root sections (1 cm long) from triploid plants were surface disinfected in 0.5% NaOCl for 30 s, transferred to water agar with 100 ppm of streptomycin sulfate, and incubated at 25°C. Slow-growing colonies were transferred to potato dextrose agar after approximately 72 h. V. dahliae was identified on the basis of morphology (3). Pathogenicity of four selected isolates was determined on the watermelon cultivars used to identify races of Fusarium oxysporum f. sp. niveum (Fon). Flasks containing 100 ml of medium (1) were inoculated with a 1-ml spore suspension at 1 × 105 spores/ml for each isolate and placed on an orbital shaker for 6 days at 100 rpm with continuous near-UV/fluorescent lighting at 25°C. Roots of approximately 40 plants of each of five watermelon cultivars (1 to 2 true-leaf stage) were trimmed to 2 cm long and root dipped for 2 min in the spore suspension (1 × 106/ml) of each isolate. Each cultivar/isolate combination and controls were transplanted into 10 pots (1.5 liter) with four plants per pot. The pots were transferred to the greenhouse where soil temperatures ranged between 15 and 25°C and were fertilized (Jack's fertilizer solution) every 7 days. Plants were rated at the end of 28 days as 1 = healthy, 2 = stunting (≤50% of controls), 3 = wilting, and 4 = dead. Initial wilting was observed within 7 to 10 days postinoculation. All four isolates caused varying degrees of vascular discoloration, stunting, wilting, and plant death. The pathogen was reisolated from symptomatic plants but not the controls. Mean disease ratings for the most virulent Texas isolate (28-040215) on ‘Black Diamond’, ‘Charleston Gray’, ‘Dixie Lee’, ‘Calhoun Gray’, and ‘PI 296341 FR’ were 2.7, 3.0, 3.0, 2.9, and 2.9, respectively. All watermelon Fon differentials were equally susceptible to V. dahliae in these studies. Historically, Verticillium wilt has been a problem in this area, which has been in cotton production for approximately 100 years. In the past decade, watermelon production has increased substantially to approximately 3,600 ha in the Texas High Plains. To our knowledge, this is the first known report of Verticillium wilt on watermelon in Texas. References: (1) R. G. Esposito and A. M. Fletcher. Arch. Biochem. Biophys. 93:369, 1961. (2) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, New York, 2002. (3) H. C. Smith. N. Z. J. Agr. Res. 8:450, 1965.

Lettuce (Lactuca sativa L.) is an important crop used for fresh and processing markets in Italy and is grown on more than 21,000 ha. During October and November of 2006, wilt symptoms were observed on field-grown lettuce, cv. Estelle, in Forlì, Emila Romagna (northeastern Italy) and on cv. Ballerina grown under plastichouses in Piedmont (northwestern Italy). Both lettuce cultivars were of a butterhead type. Affected plants were stunted and developed yellow leaves with brown or black streaks in the vascular tissue. Yellowing started from the external leaves. Discoloration was observed in the vascular tissue of roots, crown, and leaves. A fungus was consistently and readily isolated from symptomatic vascular tissue, previously disinfested in 1% sodium hypochlorite, when cultured on potato dextrose agar (PDA). Microscopic observations revealed hyaline hyphae with many ovoid, dark microsclerotia measuring 32 to 43 × 16 to 26 μm developing after 15 days of growth at 18°C in the dark. Conidiophores showed two verticils of three elements. Conidia were hyaline, elliptical, single celled, and measured 3.5 to 8.5 × 1.8 to 4.3 μm (average 5.5 × 2.5 μm). According to its morphological characteristics, the fungus was identified as Verticillium dahliae (2). Healthy, 20-day-old lettuce plants, cvs. Principessa and Maxima, both belonging to the butterhead type, were separately inoculated by root dip with a conidial suspension (106/ml) of two isolates of V. dahliae isolated, respectively, at Forlì and Torino. Noninoculated lettuce plants served as control treatments. Plants (10 per treatment) were grown in pots (10-liter vol.) in a steam-disinfested peat/perlite/sand (3:1:1 vol/vol) substrate and were maintained in a glasshouse at temperatures ranging between 17 and 22°C and relative humidity ranging between 60 and 70%. First wilt symptoms and vascular discoloration in the roots, crown, and veins developed 40 days after the artificial inoculation. Forty percent of the plants were affected in the case of cv. Maxima and 30% for cv. Principessa. Noninoculated plants remained healthy. The pathogenicity tests were repeated twice. To our knowledge, this is the first report in Italy of Verticillium wilt on lettuce. The disease has been previously reported in Greece (1) and the United States (3). Currently, Verticillium wilt of lettuce seems restricted in Italy to very few farms in the two locations; moreover, its incidence is very low (0.05%). References: (1) E. K. Ligoxigakis et al. Phytoparasitica 30:141, 2002. (2) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002. (3) G. E. Vallad et al. Plant Dis. 89:317, 2005.

The fate of methyl isothiocyanate (MITC) was studied in agricultural soils following metam sodium (MS) application in a controlled system and under field conditions as it was related to disease control. Soil samples were collected from 34 field sites in Israel with no history of MS application. The generation and dissipation curves of MITC in these soils, under controlled conditions, varied significantly among the soils, as reflected by the concentration by time (C × T) product. This value was significantly related with the mortality level of Fusarium oxysporum f. sp. radicis-lycopersici as a test organism and sand content of the soils. Seven field experiments were conducted in potato fields from 2001 to 2004. The MS treatments significantly reduced Verticillium wilt incidence and severity in five and four experiments, respectively, out of seven. Combining MS with formalin was more effective for controlling disease than MS alone in most cases. A significant relationship was found between mortality of F. oxysporum f. sp. radicis-lycopersici in soil samples to which MS was applied under controlled conditions and the incidence of Verticillium wilt disease in the field, and between CMITC × T products and the incidence of Verticillium wilt disease in the field. These tests can be used for preplant assessment of potential MS efficacy.

Lupinus polyphyllus Lindl., a perennial ornamental belonging to the Leguminosae family, is grown in gardens for flower beds and borders. During the summer of 2006, in several gardens located in the Biella Province (northern Italy), a new wilt of Lupine was observed in 20 to 30% of the plants. The vascular tissue in stems of affected plants appeared brown. These plants were stunted and developed yellow leaves with brown or black streaks in the vascular tissue. Verticillium dahliae was consistently isolated from symptomatic vascular tissue and leaves when cultured on potato dextrose agar (PDA) (3). Microscopic observations revealed hyaline hyphae, with many irregular, dark microsclerotia, ranging from 17 to 61 μm. Conidiophores showed two verticils of three elements. Conidia were hyaline, elliptical, single-celled, measuring 3.4 to 6.0 × 1.8 to 3.1 μm (average 4.5 × 2.4 μm). The ITS region (internal transcribed spacer) of rDNA was amplified using the primers ITS4/ITS6 (2) and sequenced. BLASTn analysis (1) of the 521 bp obtained showed an E-value of 0.0 with V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. EF015891. Healthy 30-day-old plants (10 per treatment) of L. polyphyllus were inoculated by root dip with a conidial suspension (0.5 × 106 CFU/ml) of V. dahliae isolated from infected plants. Ten noninoculated plants served as control treatments. All plants were transplanted into pots filled with a mix of sphagnum peat/pomix/pine bark/clay (50:20:20:10) and grown outdoors at temperatures ranging from 15 to 25°C. First wilt symptoms and vascular discoloration in the roots, crown, and veins developed within 20 days on each inoculated plant and become evident after 50 days. V. dahliae was consistently reisolated from infected plants. Noninoculated plants remained healthy. The pathogenicity test was conducted twice. To our knowledge, this is the first report of V. dahliae on L. polyphyllus in Italy. A wilt caused by V. dahliae on L. polyphyllus was observed in the Netherlands in 1925 (4). References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (3) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002. (4) J. H. H. Van der Meer. Meded. Landbouwhogesch. Wagening. 28, 1925.

Rudbeckia fulgida (common name orange coneflower) is an herbaceous perennial (Asteraceae) grown in full sun in perennial borders in gardens. At the end of the summer of 2007, in a public garden located in Turin (northern Italy), symptoms of vascular wilt and stunting were observed on approximately 80% of the plants grown in a mixed border. Initial symptoms were yellowing of external leaves and brown or black streaks in the vascular tissue of roots, crown, and leaves. A fungus was consistently and readily isolated on potato dextrose agar from symptomatic vascular tissue previously disinfested in 1% sodium hypochloride. Ovoid, dark microsclerotia, 41 to 108 μm, developed in hyaline hyphae after 10 days of growth at 23°C (12 h of light and 12 h of dark). Hyaline, elliptical, single-celled conidia, 3.2 to 7.3 × 2.1 to 3.7 (average 4.7 × 2.8) μm, developed on verticillate conidiophores. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (4). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 530 bp showed a 100% homology with the sequence of V. dahliae. The nucleotide sequence has been assigned GenBank Accession No. EU 627007. Healthy, 30-day-old R. fulgida plants were grown in a steam-disinfested mix of sphagnum peat:pomix:pine bark:clay (50:20:20:10) infested with a conidial suspension (1.5 × 106/ml) of three isolates of V. dahliae isolated from infected plants. Noninoculated plants served as controls. Plants (16 per treatment) were grown in pots (3 liter vol) and maintained in a glasshouse at temperatures between 22 and 25°C and relative humidity between 50 and 70%. First wilt symptoms and vascular discoloration in the roots, crown, and veins developed 17 days after inoculation. Noninoculated plants remained healthy. The pathogenicity tests were carried out twice. To our knowledge, this is the first report in Italy of Verticillium wilt on R. fulgida. Wilts caused by V. dahliae on R. laciniata in Poland (3) and V. albo-atrum on R. hirta in the United States (2) were previously reported. The importance and economic impact of this disease is currently limited but may increase because of the popularity of Rudbeckia spp. in private and public parks. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. F. Farr et al. Fungi on Plants and Their Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (3) B. Leski. Rocz. Nauk Roln. 253, 1974. (4) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

Klosterman, S. J., and Hayes, R. J. 2009. A soilless Verticillium wilt assay using an early flowering lettuce line. Plant Dis. 93:691-698. A soilless growth chamber assay was evaluated for rapid assessment of Verticillium wilt on lettuce (Lactuca sativa). Seedlings of the early flowering Plant Introduction (PI) 251246 were inoculated in tubes with conidial suspensions of isolates of Verticillium dahliae from lettuce or cauliflower. PI 251246 developed significant leaf and root symptoms by 21 days following inoculation with isolates of race 1 and race 2 from lettuce, but not following inoculation with an isolate from cauliflower. In contrast, leaf symptoms on the cultivar La Brillante and root symptoms on the cultivar Salinas were not easily differentiated from the symptoms observed on the noninoculated control treatments in the soilless assay, even at the highest inoculum concentration of 1 × 10 7 conidia/ml. Comparison of the soilless growth chamber assay and a soil-based greenhouse assay revealed a significantly higher proportion of PI 251246 with root vascular discoloration in the soilless assay compared with the soil-based greenhouse assay (χ 2 , 1 df = 8.9; P = 0.003). There is thus an advantage to using the soilless growth chamber assay with PI 251246. Compared with the soil-based greenhouse assay, the soilless assay reduced the time required for evaluation of Verticillium wilt symptoms on lettuce from about 90 days to 42 days.

Verticillium dahliae Kleb. causes Verticillium wilt in large numbers of crops all over the world. Common symptoms caused by the pathogen include yellowing, wilting of leaves, and discoloration in vascular tissue of the stem. In June 2007, symptoms of Verticillium wilt were observed in a grapevine (Vitis vinifera) field in the Shihezi Region of Xinjiang. To isolate the pathogen, stem segments (0.5 cm long) were surface sterilized with 1% HgCl2 for 1 min and then dipped in 70% ethanol for 10 s. The sterilized tissues were rinsed with sterile water and incubated in the dark for 7 days at 25°C on potato dextrose agar (PDA) medium. The fungus growing from the diseased tissue showed dark colonies that produced verticillate conidiophores with two to three layers with colorless, ovoid, unicellular conidia and small, black microsclerotia, which are characteristics of V. dahliae (3). To confirm its identity, ribosomal DNA fragments (regions ITS1, 5.8S rDNA, and ITS4) were PCR amplified with primer pair ITS1/ITS4 (4) and sequenced (GenBank Accession No. FJ475122). Sequential analysis revealed that the rDNA region of the fungus isolated from grapevine was identical to that of a Greek strain of V. dahliae (GenBank Accession No. AF104926). Furthermore, the specific fragment (1,500 bp) of nondefoliating pathotype of V. dahliae (1) was PCR amplified from 24 grapevine isolates of V. dahliae collected in Xinjiang, indicating that the V. dahliae pathogen from Xinjiang is a nondefoliating pathotype. To verify the causal role of the isolated fungus, pathogenicity assays were conducted on 1-year-old seedlings of the Centennial seedless cultivar. Trimmed roots were submerged in a conidial suspension (1 × 106 conidia/ml) for 30 min and sterile tap water was used as a control. The seedlings were transplanted into a pot containing 2:1 sterile mixture of peat/perlite (vol/vol). Plants were grown in a greenhouse at 25°C. Six Verticillium isolates were found to cause the same symptoms as in fields 50 days after inoculation. V. dahliae was successfully reisolated from the stems of inoculated plants. Control seedlings inoculated in sterile tap water remained healthy. Because grapevine (Vitis vinifera) is an economically important crop for fruit and winemaking material in Xinjiang, Verticillium wilt poses a threat. The disease has been previously reported in the United States (2), but to our knowledge, this is the first report from China. References: (1) E. Pérez Artés et al. Eur. J. Plant Pathol. 106:507, 2000. (2) W. C. Schnathorst and A. C. Goheen. Plant Dis. Rep. 61:909, 1977. (3) H. C. Smith. N. Z. J. Agric. Res. 8:450, 1965. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

Extensive, unprecedented wilt and mortality of the highly invasive tree-of-heaven (Ailanthus altissima) occurred recently within mixed-hardwood forests in south-central Pennsylvania. Until this study, the cause of the epidemic was unknown. Verticillium albo-atrum was consistently isolated from symptomatic Ailanthus seedlings and trees in areas having high levels of mortality, whereas V. dahliae was isolated from small scattered patches of diseased Ailanthus. Inoculations of potted Ailanthus seedlings in the greenhouse, as well as canopy trees in the field, revealed that both V. albo-atrum and V. dahliae were capable of infecting Ailanthus, but V. albo-atrum was more pathogenic. All Ailanthus seedlings and canopy trees inoculated with V. albo-atrum died within 3 months. In contrast, Ailanthus seedlings and canopy trees inoculated with V. dahliae became symptomatic, but 84% were still living 1 year following inoculation. We conclude that the major cause of Ailanthus wilt within forests of south-central Pennsylvania is V. albo-atrum. Pending further studies, including risk analysis, V. albo-atrum may be considered as a possible biocontrol agent for invasive Ailanthus.

Pistachio (Pistacia vera L.) trees in the Castilla La Mancha and Andalusia regions of central and southern Spain are grown close to olive orchards, which are often severely affected by Verticillium dahliae. During the last decade, wilt and death of one or several branches have been observed on pistachio (cv. Kerman) scions grafted on rootstock (P. terebinthus). Discoloration of vascular tissue was occasionally observed. In five surveyed orchards, incidence of affected trees was less than 2%. Wood chips with the bark removed from symptomatic trees were washed in running tap water, surface disinfested in 0.5% sodium hypochlorite for 1 min, and placed onto potato dextrose agar plates incubated at 25°C in the dark. Isolates were identified as V. dahliae on the basis of the characteristics of microsclerotia, conidiophores, and conidia. V. dahliae isolate V117 from olive was used as reference (1). The fungus was also isolated from soil in pistachio orchards using wet sieving and a modified sodium polypectate agar medium (1). Inoculum density varied from 0 to 4.73 microsclerotia per gram of soil. P. terebinthus seedlings were inoculated with two isolates of V. dahliae from pistachio trees by injecting the stems with 50 μl of a conidial suspension (107 conidia per ml) (2). Wilt symptoms of varying severity developed in 12 and 15 seedlings of the 20 pistachio seedlings inoculated with each of two isolates. No symptoms developed on the control seedlings. The pathogen was recovered from stem tissues of inoculated plants. To our knowledge, this is the first report of Verticillium wilt of pistachio in Spain. This study demonstrates the susceptibility of certain rootstocks to V. dahliae and the importance of using resistant rootstocks, such as UCBI (2), in pistachio plantations established on soils infested by V. dahliae. References: (1) F. J. López-Escudero and M. A. Blanco-López. Plant Dis. 91:1372, 2007. (2) D. P. Morgan et al. Plant Dis. 76:310, 1992.

Verticillium albo-atrum causes unprecedented wilt and mortality of the invasive tree-of-heaven (Ailanthus altissima) within forests of south-central Pennsylvania. Stem inoculation of potted Ailanthus seedlings in the greenhouse and canopy Ailanthus trees in the field with V. albo-atrum resulted in 100% mortality. Stem inoculation of understory striped maple saplings in the field also resulted in 100% mortality. However, the high susceptibility of striped maple was not observed in naturally infected stands, where only 1% of striped maple saplings exhibited Verticillium wilt. Inoculation of chestnut oak, northern red oak, red maple, sugar maple, white ash, and yellow-poplar seedlings or canopy trees with V. albo-atrum did not induce wilt symptoms. Non-Ailanthus tree species growing adjacent to dead and dying Ailanthus trees in the field were asymptomatic. Pending further studies, V. albo-atrum should be considered as a potential biocontrol agent for invasive Ailanthus.

Verticillium wilt of lettuce, caused by the soilborne pathogen Verticillium dahliae, poses a serious threat to the California lettuce industry. Knowledge of disease development and its impact on postharvest marketability would facilitate better management of the affected fields. This study investigated postharvest marketability of 22 lettuce varieties harvested from two Verticillium-infested commercial lettuce fields in Salinas and Watsonville, CA, in 2005 using a randomized complete block design. Periodic sampling to monitor disease in several crisphead varieties in the field demonstrated that root symptoms developed quickly at later stages of heading, followed by the onset of foliar symptoms as the crop reached harvest maturity. Harvested marketable heads were vacuum cooled soon after harvest to about 4°C and maintained at this temperature in commercial coolers. The impact of V. dahliae on postharvest marketability was assessed based on the percentage of heads per case deemed marketable following 1, 2, and 3 weeks of refrigerated storage. Across both field experiments, the average disease incidence and postharvest marketability ranged from 4.2 to 87.5% and from 69.4 to 100.0%, respectively, among lettuce types and varieties. The Pearson correlation analysis detected no significant relationship between disease incidence and postharvest marketability across all varieties tested (r = 0.041, P = 0.727), or within lettuce types, even though V. dahliae was recovered from 34% of the plants harvested, and recovery ranged from 0 to 73.3% for V. dahliae and from 10 to 91.7% for non-V. dahliae (V. isaacii or V. klebahnii) species. These findings demonstrate that growers can harvest lettuce from an infested field before foliar symptoms develop with negligible impact by Verticillium spp. on postharvest marketability or quality.

Solanum aethiopicum L., previously S. integrifolium Poir. (4), has been used as a rootstock for commercial, grafted eggplant production throughout Asia (3). In August 2010 and 2011, symptoms of Verticillium wilt were observed on ‘Epic’ eggplant (S. melongena L.) grafted onto S. aethiopicum at two sites with a history of the disease: one in the irrigated, dryland Columbia Basin of eastern Washington near Eltopia, and the other in maritime western Washington near Mount Vernon. Interveinal chlorosis, V-shaped necrotic lesions, and wilting were evident at both sites in both years. Each year, stems of 20 symptomatic plants from each field site were cut at the soil line to a 20-cm length, surface sterilized for 5 min in a 10% bleach solution, rinsed in tap water for 30 s, cut longitudinally, and incubated in moisture chambers for 4 weeks at room temperature in the dark. Microsclerotia that formed in the stems were typical of those produced by V. dahliae. One isolate, ‘MVEgg301’, from an infected stem at Mount Vernon, developed dark microsclerotia and verticillate conidiophores in a radiating pattern, typical of Verticillium on half-strength potato dextrose agar (1/2 PDA) medium. The internal transcribed spacer (ITS) region of rDNA amplified by PCR assay using primers ITS6 and ITS4 from mycelia sampled directly from 1/2 PDA media revealed a 100% match with ITS rDNA sequences of >50 V. dahliae accessions in GenBank (1). Pathogenicity of ‘MVEgg301’ was assessed in two tests. In both, 12 each of non-grafted and grafted (with cv. Epic) S. aethiopicum plants were inoculated with ‘MVEgg301’ by cutting approximately 5 mm off the root tips and dipping the remaining roots in a suspension of 106 conidia/ml for 5 s. Similarly, 12 each of non-grafted and grafted S. aethiopicum plants were cut and dipped similarly in sterile water as controls. Chlorosis, necrosis, and wilting were observed in 11 of the 12 inoculated, non-grafted plants and 8 of the 12 inoculated, grafted plants in Test I. The same symptoms were observed in 10 of the 12 inoculated, non-grafted plants and 10 of the 12 inoculated, grafted plants in Test II. V. dahliae was reisolated and confirmed from symptomatic, inoculated non-grafted and grafted plants using the stem assay and direct PCR assay described above. Chlorosis, necrosis, and wilting were observed in one non-grafted control plant in Test I, and two non-grafted and four grafted control plants in Test II. The symptoms were mild and likely due to nutritional deficiencies; microsclerotia were not observed in any assayed water-inoculated plant stems. Although there are several reports of V. albo-atrum infecting S. aethiopicum in the United States (2), to our knowledge, this is the first report of V. dahliae causing Verticillium wilt on this eggplant species. This finding is significant because S. aethiopicum is used as a rootstock for control of soilborne diseases like Verticillium wilt in commercial grafted eggplant production (3). References: (1) G. Calmin et al. Biotechnol. Biotechnol. Equ. 21:40, 2007. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN, 1989. (3) M. Oda. Food Fert. Technol. Ctr. Ext. Bul. 480:1, 1999. (4) PBI Solanum Project. 2012. Solanaceae Source. Accessed at http://www.nhm.ac.uk/solanaceaesource/ . Natural History Museum, London, 29 Aug. 2012.

Chinese redbud (Cercis chinensis Bunge), a member of the Fabaceae, is an important ornamental plant native to China with reported desirable medicinal effects, including stimulating blood circulation, detumescence, and detoxification (1). In October 2011, wilt symptoms of gradual leaf yellowing, wilting, scorching (marginal browning), and twig dieback were observed on plants in Yangling, Shaanxi, China. The incidence of diseased plants was about 20% in two main areas (about 20 ha in Zijingshan Park in Zhengzhou, Henan Province; and about 3,000 ha in Taiping National Forest Park in Xi'an, Shaanxi Province). Wilted leaves of diseased plant senesced and died, but defoliation was not observed. Brown discoloration was observed in vascular tissues of petioles, twigs, and stems of diseased plants, sometimes in a ring pattern. The symptoms were often restricted to the lower part of the tree or a few branches. To identify the causal agent, six twigs (each approximately 50 mm in diameter and 10 cm long) sampled from an infected tree in Yangling were rinsed in running water, surface-sterilized with 75% ethanol for 2 min, rinsed in sterilized water three times, dried, cut into 1 cm long segments, and the segments put onto potato dextrose agar (PDA) medium. A fungal isolate was recovered from diseased vascular tissues of each sample when cultured on PDA in the dark at 25°C. After 5 days, colonies changed from white to black as a result of production of microsclerotia. Microscopic observation revealed that conidiophores were hyaline and verticillate, with three to four phialides at each node. Conidia were ellipsoidal, hyaline, single-celled, and 2.5 to 7.5 × 1.25 to 4.5 μm. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (3). To prove Koch's postulates, the roots of 10 healthy, 1-year-old C. chinensis plants were each irrigated in a greenhouse with 50 ml of a conidial suspension (1.0 × 107 spores/ml) of an isolate recovered from an infected plant (2); five control plants were inoculated similarly with sterilized water. Fifteen days after inoculation, the same wilt symptoms observed on the original plants had developed on 9 of the 10 inoculated plants, whereas the control plants remained healthy. The pathogen was recovered 15 days after inoculation by isolating from petiole and stem tissues of symptomatic plants, but was not isolated from the control plants. The internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA was PCR-amplified with primers ITS1 and ITS4 (4), and sequenced. BLAST analysis of the ITS sequence (GenBank Accession No. AB735536) showed 100% homology with that of an isolate of V. dahliae (FJ572050). To our knowledge, this is the first report of Verticillium wilt on C. chinensis in China. References: (1) Y. Li et al. J. Integr. Plant Biol. 47:1021, 2005. (2) H. A. Melouk and C. E. Horner. Phytopathology 65:767, 1975. (3) G. F. Pegg and B. L. Brady. Verticillium Wilts, CABI Publishing, Oxford, UK, 2002. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.