Vascular Discoloration Research Articles

Effect of temperature on conidial germination, mycelial growth and aggressiveness of the defoliating and nondefoliating pathotypes of Verticillium dahliae from cotton in China

Three defoliating (D) pathotype isolates and five nondefoliating (ND) pathotype isolates of Verticillium dahliae from cotton in central China were tested for adaptation to various temperatures in conidial germination on water agar (10–33°C), mycelial growth on potato dextrose agar (10–33°C) and infection of cotton seedlings (Gossypium hirsutum cvs. ‘E Mian 24’ and ‘Yin Rui 361’) (25–33°C). Results showed that the D-pathotype isolates adapted better than the ND-pathotype isolates to 30°C for conidial germination and mycelial growth, although the isolates of the two pathotypes had the same optimum temperature of 25°C. Under day/night temperatures of 25/25, 27/27 and 30/25°C for 20 and 25 days, the D-pathotype isolates induced the defoliation syndrome on seedlings of the two upland cotton cultivars, whereas the ND-pathotype isolates did not induce defoliation syndrome. The values for the areas under the disease progress curve (AUDPC) and the vascular discoloration index (VDI) were higher for the D-pathotype isolates than those for the ND-pathotype isolates in each temperature treatment. Under 30/30 or 33/27°C, at least two of the three D-pathotype isolates still had higher AUDPC values and/or VDI values than all the ND-pathotype isolates on E Mian 24. Therefore, the D-pathotype isolates appear more aggressive than the ND-pathotype isolates in infection of cotton. Results also showed that the cotton cultivar Yin Rui 361 was more tolerant than the cotton cultivar E Mian 24 to infection by both pathotypes of V. dahliae. This study suggests that the D-pathotype isolates can well adapt to high temperature and heavily infect cotton under 25–30°C, and these features might be responsible for the rapid spread of this pathotype in central China.

First Report of Lasiodiplodia theobromae Causing Stem Canker of Jatropha curcas in Malaysia.

Physic nut (Jatropha curcas L.) is an important biofuel crop worldwide. Although it has been reported to be resistant to pests and diseases (1), stem cankers have been observed on this plant at several locations in Peninsular Malaysia since early February 2008. Necrotic lesions on branches appear as scars with vascular discoloration in the tissue below the lesion. The affected area is brownish and sunken in appearance. Disease incidence of these symptomatic nonwoody plants can reach up to 80% in a plantation. Forty-eight samples of symptomatic branches collected from six locations (University Farm, Setiu, Gemenceh, Pulau Carey, Port Dickson, and Kuala Selangor) were surface sterilized in 10% bleach, rinsed twice with sterile distilled water, air dried on filter paper, and plated on water agar. After 4 days, fungal colonies on the agar were transferred to potato dextrose agar (PDA) and incubated at 25°C. Twenty-seven single-spore fungal cultures obtained from all locations produced white, aerial mycelium that became dull gray after a week in culture. Pycnidia from 30-day-old pure cultures produced dark brown, oval conidia that were two celled, thin walled, and oval shape with longitudinal striations. The average size of the conidia was 23.63 × 12.72 μm with a length/width ratio of 1.86. On the basis of conidial morphology, these cultures were identified as Lasiodiplodia theobromae. To confirm the identity of the isolates, the internal transcribed spacer (ITS) region was amplified with ITS1/ITS4 primers and sequenced. The sequences were deposited in GenBank (Accession Nos. HM466951, HM466953, HM466957, GU228527, HM466959, and GU219983). Sequences from the 27 isolates were 99 to 100% identical to two L. theobromae accessions in GenBank (Nos. HM008598 and HM999905). Hence, both morphological and molecular characteristics confirmed the isolates as L. theobromae. Pathogenicity tests were performed in the glasshouse with 2-month-old J. curcas seedlings. Each plant was wound inoculated by removing the bark on a branch to a depth of 2 mm with a 10-mm cork borer. Inoculation was conducted by inserting a 10-mm-diameter PDA plug of mycelium into the wound and wrapping the inoculation site with wetted, cotton wool and Parafilm. Control plants were treated with plugs of sterile PDA. Each isolate had four replicates and two controls. After 6 days of incubation, all inoculated plants produced sunken, necrotic lesions with vascular discoloration. Leaves were wilted and yellow above the point of inoculation on branches. The control plants remained symptomless. The pathogen was successfully reisolated from lesions on inoculated branches. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts and is common in tropical and subtropical regions of the world (2,3). To our knowledge, this is the first report of stem canker associated with L. theobromae on J. curcas in Malaysia.

Enhanced resistance to Verticillium dahliae in transgenic cotton expressing an endochitinase gene from Phaseolus vulgaris

The cotton wilt disease caused by the fungus Verticillium dahliae reduces total yield and quality in cotton worldwide. Since traditional breeding has achieved only low levels of wilt resistance, we used a transgenic approach to produce wilt tolerant cotton. We produced fertile transgenic T<sub>2</sub> cotton lines by Agrobacterium-mediated transfer of an endochitinase gene from Phaseolus. Transgene integration and expression were confirmed by PCR amplification, Southern blot and Western blot analyses. Seedlings of homozygous T<sub>2</sub> plants with high levels of chitinase expression were evaluated for their tolerance to Verticillium dahliae by infecting them with a spore suspension in a greenhouse. The transgenic plants demonstrated higher degrees of tolerance to the fungus than non-transgenic plants, as measured by foliar disease symptoms, vascular discoloration and plant height. Transgenic plants were also taller than the control ones and are currently being grown in the greenhouse and will be crossed with Iranian cotton breeding lines in a future study.

The status of Botryosphaeriaceae species infecting grapevines.

Normal 0 MicrosoftInternetExplorer4 Normal 0 MicrosoftInternetExplorer4 Species in the Botryosphaeriaceae have a cosmopolitan distribution, and occur on a wide range of annual and perennial hosts including grapevines. To date, morphological and taxonomic studies, as well as analyses of nucleotide sequences of multiple genes, have allowed the identification of at least 21 different species in the Botryosphaeriaceae occurring in grapevines worldwide. Grapevine disease symptoms caused by members of this family include leaf spots, fruit rots, shoot dieback, bud necrosis, vascular discoloration of the wood, and perennial cankers, and their current status as pathogens is reviewed. Additionally, the disease name Botryosphaeria dieback is proposed here to describe the different grapevine trunk disease symptoms caused by species of Botryosphaeriaceae. Much has been written during the last decade about the association between species in the Botryosphaeriaceae and grapevine trunk diseases, which has contributed to a better understanding of the role that these fungal taxa play in grapevine diseases. Although virulence has been shown to vary between species and isolates of the same species in different countries, these fungi have become well-recognized as important grapevine pathogens worldwide. Latest and novel findings from studies conducted in different countries, on disease etiology and species distribution, epidemiology and biology are discussed. Much progress has been achieved in the development and implementation of novel diagnostic and detection techniques.Vineyard sanitation techniques, as well as chemical, biological, and cultural control strategies available at the present time to reduce the infection caused by botryosphaeriaceous fungi, are presented in this review.

Bacterial wilt of sweet potato caused by Ralstonia solanacearum in Taiwan

During the last decade, a new bacterial disease has impaired the yield of vegetable sweet potato (30–80%) in Taiwan. Infected plants developed stunting, root and stem rot, vascular discoloration and wilting. Ten bacterial isolates that caused the same symptoms in sweet potatoes after inoculation were reisolated and classified as Ralstonia solanacearum phylotype I biovar 4 based on physical and molecular analyses. Moreover, these isolates also caused wilting in convolvulaceous, solanaceaous and cruciferous plants. This report is the first of bacterial wilt of sweet potato caused by R. solanacearum in Taiwan.

Impact of laurel wilt, caused by Raffaelea lauricola, on xylem function in avocado, Persea americana

SummaryLaurel wilt, caused by Raffaelea lauricola, is a lethal, vascular disease of avocado, Persea americana. Its impact on xylem function was examined in artificially inoculated plants of the ‘Simmonds’ cultivar. Three, 7, 14, 21 and 42 days after inoculation (dai), plants were rated externally and internally for disease severity on a subjective one (asymptomatic) to 10 (dead or 100% symptomatic) scale. Stems were then cut under water, 15 cm below the inoculation point, and placed in 0.1% aqueous solutions of acid fuschin in a greenhouse. After 48 h, the percentage of functional xylem was estimated 10 and 5 cm above and below the inoculation point by quantifying acid fuschin‐stained portions of digitized stem cross‐sections. Hydraulic conductivity was determined by placing the proximal end of 5‐cm‐long stem sections, harvested between 5 and 10 cm above the inoculation point, in water and quantifying the volume of water that was drawn over time through the distal end under partial pressure (350 mm Hg). Functional xylem decreased by 3 dai, well before the development of vascular discoloration (7 dai) and wilting of foliage (14 dai). By 14 dai, extensive vascular discoloration had developed and there was a dramatic reduction in functional xylem; plants with internal disease severities of 7 or greater had <20% functional xylem. Hydraulic conductivity decreased exponentially as non‐functional xylem and disease severity increased. In plants with internal severities >7, mean flow rates of water were 0.07 ml−1 min−1 cm−2vs. 42 ml−1 min−1 cm−2 in mock‐inoculated plants. The rapid development of these changes suggests that it may be difficult to manage laurel wilt in avocado once plants are infected by R. lauricola. Better understanding of the temporal and spatial development of infection and how the host responds to infection may assist efforts to select laurel wilt‐tolerant avocado cultivars.

First report of Fusarium proliferatum causing crown and root rot of Asparagus officinalis in Turkey.

In 2011, crown and root rot symptoms appeared on 3to 8-month-old asparagus plantations in different production areas located in the Gonen district of the Balikesir province (Turkey). The symptoms included a decrease in the yield of spears and brown lesions, vascular discoloration and rot of roots, rhizomes, and stems. The disease emerged on new plantings which were established with propagation materials from infected fields. Disinfected root pieces were placed on potato dextrose agar medium and incubated at 23±1°C. Fusarium-like colonies were obtained and identified based on cultural and morphological characteristics (Leslie and Summerell, 2006). Microconidia, borne in long and short chains and false heads, were abundant, single-celled, oval, and ranged from 3.1 to 3.7×5.6 to 8.1 μm. Macroconidia were 3-5 septate, slightly curved, and ranged from 3.7 to 5.0×33.7 to 40.0 μm. The identification of the causal fungus as F. proliferatum first based on morphological data, was confirmed by speciesspecific PCR assays using the primer set CLPRO1/2 which amplified the expected 526-bp DNA fragment (Mule et al., 2004). To fulfill Koch’s postulates, pathogenicity tests were performed using a pot culture inoculation method. Sandcorn meal mixture was inoculated with plugs from F. proliferatum, incubated at 23±1°C for 10 days, then mixed with 1 kg autoclaved soil. Healthy 45-day-old asparagus seedlings were transplanted into inoculated pots and grown for 45 days at 23±1°C with 14 h photoperiod. Infected plants showed typical brown lesions in the root system, from which F. proliferatum was reisolated. This is the first report of F. proliferatum causing crown and root rot on asparagus in Turkey.

First Report of Fusarium Wilt Caused by Fusarium oxysporum f. sp. passiflorae on Passion Fruit in North America

Passiflora edulis Sims f. edulis, known as purple passion fruit, is a woody, perennial vine that is grown for its attractive two-part flower and its purple, edible fruit (4). In November 2009, passion fruit vines were collected during a regulatory nursery inspection in Santa Barbara County and submitted to the California Department of Food and Agriculture Plant Pest Diagnostics Laboratory. Nearly 100% of the plants inspected, all of which were approximately 1.25 m tall, appeared stunted, defoliated, and severely wilted. Dark brown vascular discoloration was present in the roots and lower stems of the plants. A pinkish violet Fusarium oxysporum colony containing chlamydospores, multiseptate macroconidia, and microconidia formed on monophialidic conidiophores was consistently isolated from roots and stems onto half-strength acidified potato dextrose agar (aPDA). All further experiments were done with an isolate obtained from a single conidium. A portion of the translation elongation factor gene (TEF-1α) was amplified and sequenced with primers ef1 and ef2 from our isolate (GenBank No. JF332039) (3). BLAST analysis of the 615-bp amplicon with the FUSARIUM-ID database showed 99% similarity with a F. oxysporum passion fruit isolate from Australia (NRRL 38273) (3). To confirm pathogenicity, washed roots of four-leaf stage seedlings approximately 10 cm tall were submerged in a conidial spore suspension (106 spores/ml) for 15 min. The conidial suspension was prepared by flooding 10-day-old cultures grown on aPDA medium with sterile distilled water. Seven seedlings were inoculated and planted in 10-cm2 pots and kept in a 25°C growth chamber with a 12-h photoperiod. Seven seedlings were mock inoculated with sterile water. After 3 weeks, four of the seven inoculated plants had leaves with yellow veins and discolored roots and had partially defoliated. Two of the four symptomatic plants also had brown stem cankers. F. oxysporum grew from the isolated roots and stems of all the inoculated plants. F. oxysporum did not grow from root and stem pieces from the water-dipped plants and the plants remained asymptomatic. Inoculations were repeated on plants approximately 15 cm tall with F. oxysporum growing from roots and stem pieces of all inoculated plants. Symptoms of yellow veins and root necrosis were not observed until 4 weeks after inoculation. Fusarium wilt caused by F. oxysporum f. sp. passiflorae is a significant disease of P. edulis f. edulis in Australia. The disease has also been reported in South Africa, Malaysia, Brazil, Panama, and Venezuela; but it is unclear as to whether the symptoms were caused by Fusarium wilt or Haematonectria canker (1). Banana poka (P. mollissima), P. ligularis, and P. foetida are also susceptible hosts (2). To our knowledge, this is the first report of Fusarium wilt caused by F. oxysporum f. sp. passiflorae on passion fruit in North America. Passion fruit is not commercially produced for consumption in California so the economic importance of this disease appears to be limited to nursery production and ornamental landscapes. The grower of the California nursery stated that the infected passion fruit plants had been propagated on site from seed. The source of inoculum at this nursery remains unknown.

First Report of Fusarium Wilt of Canary Island Date Palm Caused by Fusarium oxysporum f. sp. canariensis in Louisiana.

Canary Island date palm (Phoenix canariensis Hort. Ex Chabaud) is a signature palm planted in New Orleans, LA. Currently, the city has approximately 1,000 mature Canary Island date palms. During the fall of 2009, 153 palms were inspected with 27 palms exhibiting typical symptoms of Fusarium wilt. Symptoms included one-sided death and a reddish brown streak on the rachis of affected fronds and death of the leaflets. Longitudinal sections of affected fronds showed vascular discoloration. Severely infected palms were completely dead. Small pieces of diseased tissue from five palms were surface sterilized with sodium hypochlorite (0.6%) for 2 to 3 min, then rinsed in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA). Fungal colonies on PDA produced a purple pigment, and both macro- and microconidia that are typical of Fusarium oxysporum were observed under a light microscope. A single-spore culture of isolate PDC-4701 was obtained. DNA from this isolate was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and primers ef1 and ef2 were used to amplify and sequence the translation elongation factor 1-α gene (2). NCBI BLAST analysis of the 616-bp sequence resulted in 100% identity with F. oxysporum f. sp. canariensis isolates PLM-385B from Texas and PLM-511A from South Carolina (GenBank Accession Nos. HM 591538 and HM 591537, respectively). Isolate PDC-4701, grown on PDA for 2 weeks, was used to inoculate 10 9-month-old P. canariensis seedlings. An 18-gauge needle was used to inject 15 ml of a 107 conidia/ml suspension into the stem near the soil line. Each seedling was inoculated at two locations and covered with Parafilm at the inoculation sites. Ten control seedlings were injected with sterile distilled water in the same manner. Inoculated and control seedlings were maintained in a greenhouse at 28 ± 2°C. Leaves of all 10 inoculated seedlings started to wilt 3 months after inoculation. Internal vascular discoloration was observed and the pathogen was reisolated from the symptomatic seedlings. No symptoms developed on any of the 10 control seedlings. On the basis of morphology and DNA sequence data, this pathogen is identified as F. oxysporum f. sp. canariensis. Fusarium wilt of Canary Island date palm has been previously reported from California, Florida, Nevada, Texas, and South Carolina (1). To our knowledge, this is the first report of Fusarium wilt of Canary Island date palm caused by F. oxysporum f. sp. canariensis in Louisiana, extending its geographic range. The disease may adversely affect the tradition of planting Canary Island date palms in New Orleans. The sequence of isolate PDC-4701 has been submitted to the NCBI database (GenBank Accession No. JF826442) and a culture specimen has been deposited in the Fusarium Research Center culture collection (Accession No. O-2602) at the Pennsylvania State University, University Park, PA.

Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Chile.

During 2009 and 2010, a survey (n = 520) of diseased grapevines (Vitis vinifera L.) was done in vineyards located in Maipo and Colchagua valleys (33°43' to 34°36'S) in Chile. Symptoms of trunk diseases (TD) were observed on >10-year-old grapevines and consisted of short internodes, dead spurs and arms, and dieback. In cross sections, diseased arms and trunks exhibited brown, V-shaped cankers of hard consistency. Collected canker samples from cvs. Cabernet Sauvignon, Carménère, Red Globe, Syrah, and Thompson Seedless were surface sterilized in 75% ethanol for 45 s and plated onto potato dextrose agar modified with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (MPDA; Sigma-Aldrich, St. Louis, MO) for 7 days at 20°C. White-to-gray colonies with aerial mycelium growth turned dark gray after 3 to 5 days and tentatively identified as Botryosphaeriaceae. Hyphal tips of these colonies were transferred to MPDA and kept at 20°C with continuous light. After 30 days, colonies developed black, globose pycnidia with unicellular, hyaline, ellipsoidal, densely granulate, externally smooth, and thin-walled conidia that measured (16.3) 19.3 ± 2.3 (25.9) × (5.8) 7.4 ± 0.8 (9.2) μm (n = 20). Morphologically, these isolates were identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (2). Nucleotide BLAST analysis of the region ITS1-5.8S-ITS2 of rDNA of N. parvum isolates HMUC-104 and HMUC-105 (GenBank Accession Nos. JF273631 and JF273632) were amplified with ITS4 and ITS5 primers and revealed >99% similarity with the sequence of reference isolate (EU833984). Pathogenicity tests were conducted using isolates HMUC-104 and HMUC-105 on 30-day-old Carménère grapevines (n = 8) rooted in vitro by placing a 3- to 5-mm mycelial plug on the surface of the propagation medium. Additionally, detached green shoots (GS) (n = 5) and dormant canes (DC) (n = 6) 15-cm long were inoculated by placing a 3- to 5-mm mycelial plug underneath a cut aseptically made in the cortex. The GS and DC were placed in humidity chambers at 20 and 25°C, respectively. For controls, an equal number of rooted vines, in vitro vines, GS, and DC were treated with sterile agar plugs. Leaf number (LN), shoot length (SL), and root length (RL) were assessed on rooted plants in vitro after 30 days at 20°C. The extent of vascular discoloration (VD) of GS and DC were determined 15 and 45 days, respectively. N. parvum significantly (P < 0.05) reduced the LN, SL, and RL relative to the control plants. The length of VD varied from 54.86 to 55.39 mm and 14.8 to 15.48 mm in inoculated GS and DC, respectively. No VD symptoms were observed on the controls. N. parvum was reisolated from 100% of the inoculated in vitro plants, GS, and DC, completing Koch's postulates. N. parvum has been documented as a canker pathogen on V. vinifera and is known to contribute to the decline of grapevines. To our knowledge, this is the first report of N. parvum causing bot canker on grapevines in Chile, but has previously been reported in Australia, Spain, and the United States. Of 520 diseased plants in this study, 10 to 15% prevalence was estimated for TD and almost 2% prevalence was associated to N. parvum. Other Botryosphaeriaceae spp. were isolated with N. parvum from grapevine TD in Chilean vineyards (1,3,4).

Verticillium dahliae's VdNEP acts both as a plant defence elicitor and a pathogenicity factor in the interaction with Helianthus annuus

The soil-borne fungus Verticillium dahliae Kleb. causes a serious wilt disease in many economic crops worldwide, including sunflower. We investigated the role of V. dahliae's necrosis and ethylene-inducing proteins (VdNEP) in the interaction with sunflower. We inoculated two highly and two weakly aggressive V. dahliae isolates onto moderately resistant and susceptible sunflower hybrids. We also synthesized VdNEP protein in vitro and infiltrated it into sunflower control plants to compare its effects with those of the fungus. VdNEP induced wilting symptoms, i.e. chlorosis, necrosis and vascular discoloration but also triggered host defence responses, i.e. hypersensitive response in Nicotiana benthamiana leaves and sunflower cotyledons. It activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves, as well as pathogenesis-related genes (Ha-PR-3 and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and those encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO. The latter suggested that more than one signalling pathway may be involved in the V. dahliae-sunflower interaction. Two other genes (Ha-PAL and Ha-NML1), related to the salicylic acid pathway, were slightly downregulated by VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defences. The phenotypic changes induced by VdNEP indicate that this protein acts both as a defence elicitor and a pathogenicity factor.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Coleus verschaffeltii in Italy.

Coleus verschaffeltii Lem. (synonym C. blumei Benth., Plectranthus scutellaroides (L.) R. Br., and Solenostemon scutellarioides (L.) Codd), a perennial plant belonging to the Lamiaceae family, is used as a bedding plant for public gardens. The most popular cultivars produce speckled leaves of various colors. In October 2010, severe outbreaks of a previously unknown wilt were observed in a public garden at Torino (northern Italy) on 50 8-month-old plants. Plants were sprinkle irrigated. Initial symptoms were withering of leaves starting from the collar and brown streaks in the vascular tissue of roots, crown, and stem. Subsequently, infected tissues wilted and plants became stunted. Early leaf drop was observed and plants appeared bare, keeping few leaves only at the end of stems. Infected plants did not die but they lost the original ornamental aspect. Seventy percent of the plants were affected. Stems of 10 plants were disinfected with 1% sodium hypochlorite. Cross-sections through symptomatic vascular tissues were plated on potato dextrose agar amended with 25 ppm of streptomycin sulfate. After 10 days at 20 to 23°C, a fungus was consistently recovered from 90% of stems. Irregular, black microsclerotia, 29 to 76 × 14 to 52 (average 49 × 28) μm, developed in hyaline hyphae after 15 days of growth. Hyaline, elliptical, single-celled conidia, 3.9 to 7.2 × 1.7 to 2.8 (average 5.1 × 2.2) μm, developed on verticillate conidiophores with three phialides at each node. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (3). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (4) and sequenced. BLASTn analysis (1) of the 491-bp segment showed a 99% homology with the sequence of V. dahliae (Accession No. GU461634). The ITS nucleotide sequence of our isolate has been assigned the GenBank Accession No. JF704205. Pathogenicity tests were performed twice using 45-day-old plants obtained from seeds of C. verschaffeltii grown in 1-liter pots containing a 50:20:20:10 steamed mix of peat moss/pumice/pine bark/clay. Roots of 10 healthy plants were immersed in a conidial suspension (1.7 × 107 ml-1) of one culture of V. dahliae isolated from infected plants. Ten plants immersed in sterile water served as controls. Plants were maintained in a glasshouse at daily average temperatures between 20 and 28°C and relative humidity between 50 and 80%. First wilt symptoms and vascular discoloration in the roots, crown, and stems developed 20 days after inoculation. V. dahliae was consistently reisolated from infected vascular tissues of crown and stems of symptomatic plants. Noninoculated plants remained healthy. To our knowledge, this is the first report of Verticillium wilt on C. verschaffeltii in Italy. Verticillium wilt had been previously reported on S. scutellaroides in the United States (2). At this time, the economic importance of Verticillium wilt on C. verschaffeltii in Italy is limited.

First Report of Basal Stem Rot of Apple Cactus (Cereus peruvianus monstruosus) Caused by Fusarium oxysporum in Italy.

During the summer of 2010, 20% of 7,000 4-month-old plants of apple cactus (Cereus peruvianus monstruosus) showed symptoms of a basal stem rot in a commercial nursery located in Liguria (northern Italy). Affected plants showed yellow orange-to-pale brown color from the crown level to the stem apex and a water-soaked rot was observed on the stem starting from the base. Brown discoloration was observed in the vascular system. Eventually stems bent, plants collapsed and died, and affected tissues dried out. A Fusarium sp. was consistently and readily isolated from symptomatic tissue on Komada selective medium. Isolates were purified and subcultured on potato dextrose agar (PDA). Single-spore cultures on PDA, Spezieller Nährstoffarmer agar (SNA) (3), and carnation leaf-piece agar (CLA) (2) were incubated at 26 ± 1°C (12-h fluorescent light, 12-h dark). On PDA, cultures produced a thick growth of white-to-pink mycelium and pale pink pigments in the agar. On SNA, cultures produced short monophialides with unicellular, ovoid-elliptical microconidia measuring 4.3 to 8.2 × 2.3 to 3.8 (average 6.0 × 2.8) μm. Chlamydospores were abundant, single or paired, terminal and intercalary, rough walled, and 6 to 8 μm in diameter. On CLA, cultures produced orange sporodochia with macroconidia that were 3 to 4 septate, nearly straight with a foot-shaped basal cell and a short apical cell, and measured 31.1 to 51.5 × 4.4 to 3.5 (average 43.2 × 3.8) μm. Such characteristics are typical of Fusarium oxysporum (3). Amplification of the ITS (internal transcribed spacer) of the rDNA using primers ITS1/ITS4 (4) yielded a 498-bp band. Sequencing and BLASTn analysis of this band showed an E-value of 0.0 with F. oxysporum. The nucleotide sequence has been assigned GenBank Accession No. JF422071. To confirm pathogenicity, five 6-month-old healthy plants of C. peruvianus monstruosus were inoculated by dipping roots in a conidial suspension (2.4 × 106 CFU/ml) of F. oxysporum isolated from affected plants. Inoculum was obtained from pure cultures of three single-spore isolates grown for 10 days on casein hydrolysate liquid medium. Roots were not wounded before the inoculation. Plants were transplanted into pots filled with steam-sterilized substrate (sphagnum peat/perlite/pine bark/clay 50:20:20:10). Five noninoculated plants served as a control. Plants were placed in a climatic chamber at 25 ± 1°C (12-h fluorescent light, 12 h-dark). Basal stem rot and vascular discoloration in the crown and stem developed within 30 days on each inoculated plant. Noninoculated plants remained healthy. F. oxysporum was consistently isolated from symptomatic plants. The pathogenicity test was conducted twice. F. oxysporum has been reported on Cereus spp. in the United States (1). To our knowledge, this is the first report of F. oxysporum on C. peruvianus monstruosus in Italy as well as in Europe. Currently, this disease is present in a few nurseries in Liguria.

First Report of Fusarium Wilt on Philotheca myoporoides Caused by Fusarium oxysporum in Italy.

Philotheca myoporoides (DC.) M.J. Bayly (previously known as Eriostemon myoporoides), commonly called long-leaf waxflower and native to eastern Australia (Rutaceae family), is a hardy compact shrub or small tree occurring in subtropical to cool temperate regions. P. myoporoides is cultivated in Sicily (Italy) for its ornamental appeal. During April of 2010, a widespread wilting was observed on approximately 80% of 2,000 1-year-old, potted long-leaf waxflower plants grown in a commercial nursery near Catania (eastern Sicily, Italy). Internally, symptomatic plants had conspicuous vascular brown discoloration from the crown to the canopy. Diseased crown and stem tissues of 20 plants were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with white or light purple aerial mycelia and violet pigmentation on the underside of the cultures developed after 9 days. On carnation leaf agar, 20 single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on one representative isolate (ITEM 13490) by analyzing sequences of the benA gene (coding β-tubulin protein) and CaM gene (coding calmodulin protein) using the primers reported by O'Donnell et al. (1). The benA gene sequences of ITEM 13490 (GenBank No. FR828825) exhibited an identity of 100% to Fusarium oxysporum f. sp. radicis-lycopersici strain ATCC 52429 (GenBank No. DQ092480). CaM gene sequences of ITEM 13490 (GenBank No. FR828826) exhibited an identity of 99.6% to F. oxysporum strain ITEM 2367 (GenBank No. AJ560774). Morphological characteristics of the 20 isolates, as well as the PCR assay on a representative strain, identified the isolates associated with disease symptoms as F. oxysporum Schlechtend.:Fr. A pathogenicity test was performed by placing two 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 2-month-old cuttings of P. myoporoides. Thirty plants were inoculated with strain ITEM 13490 and the same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 25 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. First symptoms, which were identical to those observed in the nursery, developed on one plant 15 days after inoculation. Wilting was detected on all plants after 30 days. Control plants remained symptomless. F. oxysporum was successfully reisolated from symptomatic crown and stem tissues and identified as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum causing disease of P. myoporoides worldwide. Moreover, this pathogen was recently reported in the same nursery on Eremophila sp. (2), confirming the presence of Fusarium wilt as a potential threat to ornamental plant production in this area, and necessitates the innovation and development of disinfection methods for alveolar trays, greenhouses, and various propagation materials to reduce future disease outbreaks.

First Report of Cryptovalsa ampelina and Eutypella leprosa Associated with Grapevine Trunk Diseases in Chile.

Trunk diseases (TD) of grapevines (Vitis vinifera L.) have increased considerably in Chile with an incidence of more than 25% found in ≥7-year-old vineyards. Only species of Botryosphaeriaceae, Phaeoacremonium, and Phaeomoniella were associated with TD in Chile (1,2). From 2009 to 2010, isolations were made from the grapevines 'Cabernet Sauvignon', 'Carmenere', 'Flame Seedless', and 'Pinot Noir' collected in central Chile (33°27' to 34°39'S, 71°17' to 71°33'W). These grapevines showed cankers and vascular necrosis of trunks, arms, and spurs along with a general decline and dieback. Isolations were performed in potato dextrose agar (PDA) plus 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (Sigma-Aldrich, St. Louis, MO), for 14 days at 20°C. On the basis of colony morphology and conidia production, two Libertella-like species were obtained in 26 (7.8%) of 335 trunk samples. On the basis of the internal transcribed spacer region (ITS4 and ITS5) of rDNA, Cryptovalsa ampelina (Nitschke) Fuckel (GenBank Accession Nos. HQ694976 and HQ694977), and Eutypella leprosa (Persoon) Berlese (HQ694974 and HQ694975) were identified, showing 98 to 100% similarity with the sequences of C. ampelina (GQ293913) and E. leprosa (AJ302463.1). C. ampelina produced white-to-creamy, smooth colonies with a creamy underside. Colonies of E. leprosa were white-to-gray with a white underside. Orange conidial masses were exuded after 30 days at 20°C. Conidia on PDA (n = 20) were unicellular, hyaline, filiform, slightly curved, and (19.8) 23.4 ± 2.6 (28.3) × (1.1) 1.4 ± 0.2 (1.8) μm and (19.2) 23.9 ± 3.0 (27.6) × (1.0) 1.2 ± 0.1 (1.5) μm for E. leprosa and C. ampelina, respectively. Stromatic perithecia of C. ampelina, embedded in the bark, were observed in dead pruning residues of infected vines (4). Pathogenicity tests were conducted with two isolates of each species, on 30-day-old 'Carmenere', rooted in vitro (n = 12), that were inoculated by placing a 5-mm agar plug on the surface of the propagation medium. Additionally, 15 cm long pieces (n = 5) of 1-year-old canes from 'Carmenere', 'Chardonnay', and 'Red Globe' were inoculated by placing a 5-mm agar plug underneath a cut aseptically made in each cane. An equal number of noninoculated plants and canes, but treated with sterile agar plugs, were used as controls. Leaf number (LN), shoot length (SL), and root length (RL) were assessed on plants in vitro after 28 days at 20°C. The extent of vascular discoloration (VD) obtained in canes was determined after 45 days in humid chambers at 20°C. One-way analysis of variance was performed and mean differences were studied by Tukey's test. C. ampelina and E. leprosa significantly (P < 0.05) reduced the LN, SL, and RL relative to the control plants. They also caused a VD of 10.1, 11.6, and 9.8 mm and 11.2, 13.4, and 10.0 mm in 'Carmenere', 'Chardonnay', and 'Red Globe', respectively. No symptoms were observed on the control canes. C. ampelina (100%) and E. leprosa (75%) were reisolated from inoculated plants and canes. To our knowledge, this is the first report of C. ampelina and E. leprosa on grapevines in Chile. However, their relative importance as causal agent of trunk disease remains to be determined. C. ampelina and E. leprosa have been associated with grapevine cankers in the United States and Spain (3,4).

First Report of Fusarium Wilt of Chicory (Cichorium intybus) Caused by Fusarium oxysporum in Italy.

In the summer of 2009, a wilt of chicory was observed on 25 to 30% of 30-day-old Cichorium intybus L. cv. Clio plants grown outdoors on a commercial farm in Piedmont (northern Italy). Affected plants were chlorotic and stunted with poorly developed root systems compared with healthy plants. Black streaks were observed in the stem and proximal part of the leaf vascular system in wilted plants. Fusarium oxysporum Schltdl. was isolated from symptomatic vascular tissue on a Fusarium-selective medium (1) from 80% of samples. Grown on potato dextrose agar (PDA) for 4 days at 23°C, the colonies, initially white and later pale pink, produced hyaline microconidia that were oval-elliptical and cylindrical in shape measuring 5.6 to 14.9 (average 10.2) × 2.1 to 4.5 (3.0) μm, borne on short monophialides measuring 8.2 to 16.1 (average 13.2) × 2.1 to 4.2 (3.3) μm. Macroconidia were slightly curved, three-septate, with a slightly hooked apical cell and a foot-shaped basal cell measuring 24.9 to 41.6 (average 32.2) × 3.2 to 5.2 (4.3) μm. Chlamydospores were both terminally and intercalary, solitary but also in short chains (2 to 4 elements) measuring 21.1 to 41.0 (average 27.2) μm (2). The internal transcribed spacer (ITS) rDNA region was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 527-bp amplicon (GenBank Accession No. HQ644423) obtained had 98% sequence identity with F. oxysporum (GenBank Accession No. FJ605247). The translation elongation factor-1α (EF-1α) gene was amplified using primers EF-1/EF-2 and sequenced (GenBank Accession No. GU564259). The 663-bp fragment had 99% sequence identity with F. oxysporum (GenBank Accession Nos. EU313540, EU313539, and DQ837696). Pathogenicity tests were conducted on 15-day-old chicory plants from two cultivars (Clio and Katia). Thirty-five plants per cultivar were inoculated by dipping their roots in a 1 × 106 CFU/ml suspension of isolate FusCic45B recovered from wilted chicory. Inoculated and noninoculated plants were transplanted into five pots filled with 10 liters of steamed mix (peat/perlite/sand, 60:20:20 vol/vol) and were maintained in a glasshouse at 25 to 27°C. Wilt symptoms and vascular discoloration of the roots, crown, and veins developed 15 days after inoculation on all inoculated plants. Plants of cv. Clio were more susceptible. F. oxysporum was always reisolated from infected plants using the Fusarium-selective medium. All noninoculated plants remained healthy. The pathogenicity test was conducted twice. To our knowledge, this is the first report of wilt caused by F. oxysporum on chicory, C. intybus, in Italy as well as worldwide.

Isolation and characterization of antagonistic bacteria against fusarium wilt and induction of defense related enzymes in banana

Fusarium wilt caused by Fusarium oxysporum is considered a fatal disease to bananas.Chemical control of Fusarium wilt in bananas has received little efficiency. Alternatively, biological control is a viable strategy against soil-borne diseases. The objectives of this research were to isolate antagonistic bacteria and investigate the possible mechanisms against Fusarium wilt. An antagonistic Bacillus strain, termed KY-21 against F. oxysporumf. sp. Cubense was isolated from the soil of banana’s rhizosphere by dual culture. The strain was identified as Bacillus subtilis according to the characteristics of its morphology and by homology analysis of its 16S rDNA sequence. Mycelium growth of the pathogen was seriously inhibited after treatment with the fermentation filtrate of KY-21. Observation revealed that the tips of the hypha were deformed into spherical structures that were remarkably constricted. The strain KY-21 displayed a good ability to colonize and can transfer into banana tissues. The activities of polyphenol oxidase (PPO) and peroxidase (POD) in bananas showed an increase after inoculated with KY-21, compared to control.In vivo biocontrol assays showed a significant reduction in wilt index and vascular discoloration. These results indicated that the antagonistic mechanisms against Fusariumwilt were involved in the inhibition of mycelial growth and the improvement in activity of defense related enzymes. Key words: Fusarium oxysporum f. sp. Cubense, antagonistic bacteria, screen,colonization, enzyme activity.

Efficacy of Four Soil Treatments Against Fusarium oxysporum f. sp. vasinfectum Race 4 on Cotton

Fusarium wilt, caused by race 4 of Fusarium oxysporum f. sp. vasinfectum, is a critically important disease problem in California cotton (Pima, Gossypium barbadense; Upland, G. hirsutum). Because few cultivars with resistance to race 4 are available, alternative management strategies for this disease are needed. Four soil treatments (50:50 methyl-bromide + chloropicrin as a positive control; 60:40 chloropicrin + 1,3-dichloropropene; 6 weeks of solarization; and metam-sodium) were evaluated for efficacy against race 4 in a naturally infested, heavy clay soil. Treatments were evaluated based on plant mortality, height, number of mainstem nodes, vascular discoloration ratings, and soil counts of F. oxysporum. Two cultivars each of Pima and Upland cotton varying in resistance to race 4 were used. Plant mortality was lowest in methyl-bromide + chloropicrin, solarization, and chloropicrin + 1,3-dichloropropene treatments, and highest in the nontreated and metam-sodium treatments. Although most plant mortality occurred within 5 weeks after planting, substantial mortality of the susceptible Pima cultivar DP 744 accumulated for up to 10 weeks. Seven to eight weeks after planting, plants in methyl-bromide + chloropicrin and chloropicrin + 1,3-dichloropropene treatments were taller and had more mainstem nodes than in other treatments. Vascular discoloration was reduced in methyl-bromide + chloropicrin and solarization treatments compared with the nontreated control, metam-sodium, and chloropicrin + 1,3-dichloropropene treatments. Soil counts of F. oxysporum were significantly reduced only in the methylbromide + chloropicrin, chloropicrin + 1,3-dichloropropene, and solarization treatments. Six weeks of solarization and 60:40 chloropicrin + 1,3-dichloropropene (295 liters a.i./ha) proved effective for reducing Fusarium wilt of cotton in heavy clay soil.

First Report of Fusarium Wilt on Eremophila spp. Caused by Fusarium oxysporum in Italy.

Eremophila spp. (Myoporaceae family), endemic to Australia, are evergreen shrubs or small trees occurring in arid, semi-arid, tropical, or temperate regions. In Europe, Eremophila spp. are grown for their horticultural appeal. During 2009 and 2010, extensive wilting was observed on 2-month to 1-year-old potted plants of Eremophila laanii F. Muell., E. glabra subsp. carnosa Chinnock, and E. maculata (Ker Gawl.) F. Muell. grown in a commercial nursery near Catania (southern Italy). Internally, symptomatic plants had conspicuous vascular discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with purple mycelia and violet reverse colors developed after 9 days. On carnation leaf agar, single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregated chlamydospores. A PCR assay was conducted on two representative isolates (ITEM 12591 and ITEM 12592) by analyzing sequences of the partial CaM gene (coding calmodulin protein) and benA (coding beta-tubulin protein) using the primers as reported by O'Donnell et al. (1). Calmodulin sequences of ITEM 12951 and ITEM 12952 isolates (GenBank Nos. FR671157 and FR671158) exhibited 99.8 and 99.5% identity with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), respectively, and had 99.5% homology between them. BenA gene sequences of ITEM 12951 (GenBank No. FR671426) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain CC-612-3 (GenBank No. AY714092.1), and benA gene sequences of ITEM 12952 (GenBank No. FR671427) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain LA 140 (GenBank No. FJ466740.1), whereas the homology between the two strains is 99.5%. Morphological characteristics, as well as CaM and benA sequences, identified the isolates as F. oxysporum Schlechtend:Fr. Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 3-month-old cuttings of E. laanii, E. glabra subsp. carnosa, and E. maculata. Twenty plants for each species were inoculated with each isolate. The same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. Symptoms identical to those observed in the nursery developed 20 days after inoculation with both strains. Crown and stem discoloration was detected in all inoculated plants after 45 days. Wilting was detected on 15% of plants. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously above. To our knowledge, this is the first report of F. oxysporum causing disease of Eremophila spp. worldwide. Reference: (1) K. O'Donnell et al. Mycoscience 41:61, 2000.

First Report on the Occurrence of a Virulent Strain of Fusarium Wilt Pathogen (Race-1) Infecting Cavendish (AAA) Group of Bananas in India

Banana wilt disease caused by Fusarium oxysporum f. sp. cubense is one of the most significant threats to banana production worldwide. Strains of F. oxysporum f. sp. cubense have been grouped into race-1, -2, or -4 on the basis of differential virulence among different genotypes of banana. In India, though the disease is reported among susceptible varieties of races 1 and 2, the disease is not reported from Cavendish cultivars, which are the differential host to race-4. Recent surveys of the Cumbum areas (Theni District, Tamil Nadu) revealed symptoms (e.g., yellowing and drooping of leaves around the pseudostem and longitudinal splitting of pseudostem) on cv. Grand Naine (Cavendish group - AAA). F. oxysporum f. sp. cubense was recovered and single-spore isolates had characteristic white-to-purple aerial mycelia producing single-celled, oval microconidia in false heads on branched monophialides and sickle-shaped macroconidia with an attenuated apical cell and a foot-shaped basal cell. Pathogenicity was demonstrated on cv. Grand Naine by inoculation with sand maize meal inoculum (20 g per pot containing 106 spores per g). Vegetative compatibility, using 33 nit-M testers of all known vegetative compatibility groups (2), showed that nit-1 mutants generated from a wild strain of F. oxysporum f. sp. cubense isolated from cv. Grand Naine formed robust heterokaryons with nit-M tester 0124 of the Department of Employment, Economic Development and Innovation, Brisbane, Australia and also with nit-M tester obtained from an isolate of F. oxysporum f. sp. cubense from Karpuravalli (Pisang Awak-ABB). Further characterization of this new Cavendish strain was studied on the basis of volatile odor production (3) using VCGs 0125 for race-1 ('inodoratum group') and 0120 for race 4 ('odoratum group') as positive controls and sterile medium as a negative control. This new F. oxysporum f. sp. cubense strain of Cavendish belonged to 'inodoratum' group of F. oxysporum f. sp. cubense. Pathogenicity was demonstrated on potted plants (10 per cultivar) of cvs. Rasthali (Silk-AAB), Karpuravalli (Pisang Awak-ABB), Ney Poovan (AB), Poovan (Mysore-AAB), Red Banana (AAA), Nendran (French plantain-AAB), Monthan (ABB), and Grand Naine (Cavendish-AAA) by inoculation with sand maize meal inoculum (20 g per pot containing 106 spores per g) in three replicate experiments. Plants were uprooted 2 months postinoculation and disease severity was estimated by rating internal vascular discoloration in the corm (1). The result showed that all cultivars, except Red Banana and Nendran, had the highest rating for disease severity, 6. To our knowledge, this is the first report of a virulent strain of F. oxysporum f. sp. cubense VCG 0124 of race-1on Cavendish banana.