Diseased Strawberry Plants Research Articles

Aerial dispersion of Xanthomonas fragariae during trimming leaves of angular leaf spot diseased strawberry propagation plants

SummaryThe bacterium Xanthomonas fragariae is the causative agent of angular leaf spot (ALS) of strawberry, a Regulated Non-Quarantine Pest in Europe (EPPO A2) for plant propagation material. Field experiments were conducted to explore if X. fragariae is dispersed through the air during trimming ALS-diseased strawberry plants with dry or wetted leaves. Trimming the leaves led to dissemination of leaf fragments to the nearby surroundings. A sharp decrease in the amount of leaf fragments within the first 5 m distance downwind from the strawberry plants was found. Furthermore, air quality monitors demonstrated that during trimming 0.5 – 10 μm sized particles were ejected into the air, resulting in short periods with increased particle densities 45 cm above ground level, which could be detected at least 50 m downwind. At this height X. fragariae was detected by means of air samplers, as evidenced with a combination of dilution-plating and TaqMan assays, at 25 m distance downwind from ALS-diseased plants. A sharp decrease in the density of X. fragariae colony forming units (cfu) within the first 10 m distance from the source plants was found. The densities of X. fragariae cfu were strongly associated with the particle densities. Results indicate that during trimming leaves of strawberry propagation crops with ALS-diseased plants there is a considerable risk of deposition of airborne X. fragariae inoculum on nearby nursery beds. Whether this airborne inoculum can result in infections is discussed.

A Semi-Selective Medium to Evaluate Over-Summering Survival of Neopestalotiopsis sp. in Florida Strawberry Fields.

Devastating disease outbreaks with leaf spot symptoms and fruit rot caused by a fungus identified as a cryptic species of Neopestalotiopsis have generated concern in the Florida strawberry industry. Some Neopestalotiopsis species are saprobes in soil and plant debris, but the ability of the new Neopestalotiopsis sp. to survive in strawberry debris was unknown. Therefore, the objectives of this study were to develop a semiselective medium for isolation of Neopestalotiopsis spp. and to evaluate Neopestalotiopsis sp. survival in soil and strawberry plant debris over the summer in Florida. The Neopestalotiopsis semiselective medium (NSM) inhibited growth of most fungal species pathogenic to strawberry in Florida, except Neopestalotiopsis rosae, Neopestalotiopsis sp., and Phomopsis obscurans, in addition to Penicillium spp., which are nonpathogenic. However, Neopestalotiopsis species, P. obscurans, and Penicillium spp. could be distinguished in the medium through morphological characteristics. Soil samples arbitrarily collected from six commercial fields toward the end of the season (May) and before (July) and after (September) preplant soil fumigation in the following season were processed with NSM, and Neopestalotiopsis spp. populations were calculated as CFU per gram of soil. CFU ranged from 48.3 to 2,410.8 at the end of the season and from 1.7 to 630.8 before soil fumigation, but Neopestalotiopsis spp. were not recovered after soil treatment. However, 1.7 to 25 CFU were obtained from the nontreated areas in the row middles. Neopestalotiopsis sp. survival was also evaluated on diseased strawberry plants (leaves and crowns) exposed to the environment for 17 months. On leaves, Neopestalotiopsis spp. were recovered for 6 months before leaf decomposition, whereas the number of CFU on crowns declined monthly, but colonies were still recovered during the final evaluation months. A high-resolution melting analysis confirmed most of the colonies from soil and crowns were the new Neopestalotiopsis sp. Our results show Neopestalotiopsis sp. can survive in soil and strawberry debris under Florida summer conditions and may serve as a source of inoculum for the subsequent season.

Rhizosphere microbial ecological characteristics of strawberry root rot.

Strawberry (Fragaria × ananassa Duch.) holds a preeminent position among small fruits globally due to its delectable fruits and significant economic value. However, strawberry cultivation is hampered by various plant diseases, hindering the sustainable development of the strawberry industry. The occurrence of plant diseases is closely linked to imbalance in rhizosphere microbial community structure. In the present study, a systematic analysis of the differences and correlations among non-culturable microorganisms, cultivable microbial communities, and soil nutrients in rhizosphere soil, root surface soil, and non-rhizosphere soil of healthy and diseased strawberry plants affected by root rot was conducted. The goal was to explore the relationship between strawberry root rot occurrence and rhizosphere microbial community structure. According to the results, strawberry root rot altered microbial community diversity, influenced fungal community composition in strawberry roots, reduced microbial interaction network stability, and enriched more endophytic-phytopathogenic bacteria and saprophytic bacteria. In addition, the number of bacteria isolated from the root surface soil of diseased plants was significantly higher than that of healthy plants. In summary, the diseased strawberry plants changed microbial community diversity, fungal species composition, and enriched functional microorganisms significantly, in addition to reshaping the microbial co-occurrence network. The results provide a theoretical basis for revealing the microecological mechanism of strawberry root rot and the ecological prevention and control of strawberry root rot from a microbial ecology perspective.

Sporodochia Formed by Fusarium oxysporum f. sp. fragariae Produce Airborne Conidia and Are Ubiquitous on Diseased Strawberry Plants in California.

Sporodochia are dense masses of fungal hyphae bearing asexual conidia. For Fusarium oxysporum, sporodochia are known to produce airborne conidia and enhance the dissemination of this otherwise soilborne pathogen. Sporodochia are small and transient, and they are documented for only a few formae speciales of F. oxysporum. This study reports airborne conidia and sporodochia produced by F. oxysporum f. sp. fragariae, the cause of Fusarium wilt of strawberry, in the Monterey Bay region of California. Sporodochia were discovered in 21 of 24 Fusarium wilt-diseased fields surveyed for this study and were readily observed on most symptomatic plants in these fields. Only necrotic tissues bore sporodochia, and they were most frequently observed on petioles and peduncles. Sporodochia covered significantly greater lengths of peduncles than petioles, extending from the base of the plant toward the upper part of the canopy. A stolon hosted the longest stretch of sporodochial growth, found covering the stolon's entire 35-cm length and the base of the daughter plant. Macroconidia were produced by all sporodochia samples, and we did not find microconidia on any samples. An initial series of experiments confirmed the potential for conidia produced by sporodochia to disperse with wind over short distances. The prevalence of sporodochia producing airborne spores of F. oxysporum f. sp. fragariae has great importance for disease management and biosecurity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Bacterial Wilt Caused by Enterobacter mori of Strawberry in Beijing, China.

Strawberry (Fragaria × ananassa) is an economically important crop in China, and a crucial part of urban agriculture in Beijing. In November 2020, wilt symptoms were observed in strawberry seedlings in several greenhouses in the Pinggu District of Beijing city (40.14° N; 117.12° E). The average disease incidence was 20%. Water-soaked lesions appeared along the veins of diseased strawberry leaves and bacterial ooze was also present on severely affected leaves. Bisected crowns had a reddish-brown discoloration in the xylem which later turned black. Three diseased strawberry seedlings were collected for pathogen identification. Isolations were conducted from stem, crown, leaf, and roots of diseased strawberry plants. Samples were surface sterilized by immersion in 70% ethanol for 30 s and rinsed three times with sterile distilled water, before being placed on potato dextrose agar (PDA) medium and incubated at 28℃. Several bacterial colonies grew on the medium after 24 h. Colonies were then purified on Lysogeny broth (LB) agar plates using the streak plate method. Twenty-nine isolates were obtained from 36 diseased tissue samples, which were from stem(10), crown(12), leaf(2) and roots(5) separately. All isolates appeared white, round, opaque and smooth on LB plates. To identify the isolates, genomic DNA was extracted from nine purified bacterial colonies (CM1 to CM9). The fragments of atpD, gyrB, infB and rpoB gene were amplified and sequenced with primers atpD 01-F/ atpD 02-R, gyrB 01-F/ gyrB 02-R, and infB 01-F/ infB 02-R (Brady et al. 2008) and RpoB-F/ RpoB-R (Mollet et al. 1997), respectively. All atpD, gyrB, infB and rpoB sequences belonging to the isolates were identical. The sequences of atpD, gyrB, infB and rpoB gene of isolates CM1 and CM3 were deposited in GenBank under accession numbers ON055247, ON055248, ON055249, ON055250, ON055251, ON055252, OL771192 and OL771193. BLAST searches were conducted with the sequences of atpD, gyrB, infB and rpoB. The atpD, gyrB, infB and rpoB sequences of the obtained isolate showed 99.53%, 99.06%, 99.19% and 99.80% identity with the corresponding sequences of Enterobacter mori strains, respectively. Phylogenetic analysis was performed using the maximum likelihood (ML) method with the CIPRES Science Gateway platform (http://www.phylo.org/) based on the combined atpD, gyrB, infB and rpoB sequences (Brady et al. 2013; Palmer et al. 2018). In the phylogenetic tree, the isolates were clustered together with E. mori strain LMG 25706. To confirm the pathogenicity, 200 μL of bacterial suspensions (108 CFU/mL) of the two isolates were injected into the crown of six healthy Fragaria × ananassa cv. Bennihope strawberry seedlings respectively with 1 mL sterilized syringe, and the control seedlings were injected with sterile water. The seedlings were kept in a moist chamber (28°C, 16-h light and 8-h dark period) for 2 days. Then all the seedlings were transferred to the greenhouse with conditions similar to those where the diseased plants were collected. Forty days after inoculation, old leaves started to wilt and leaf midvein necrosis, along with xylem discoloration, was observed in inoculated plants. No symptoms were observed in the control group. Pathogenicity tests were conducted three times with similar results. The bacteria were re-isolated from the symptomatic diseased strawberry plants and confirmed as E. mori by morphological and sequence analyses as above, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of strawberry bacterial wilt caused by E. mori. Due to the significant crop loss from this disease, more research is needed in epidemiology and disease management.

PHYLOGENETIC ANALYSIS OF FUSARIUM OXYSPORUMF STRAINS ISOLATED FROM STRAWBERRY CROPSFRAGARIA ANANASSADUCH IN THE PROVINCE OF PICHINCHA (ECUADOR)

Objective: Strawberry cultivation has acquired great importance for consumption, promoting the increase of its production in Ecuador. However, the process of importing plant material from producing countries in order to improve domestic production has contributed to the dissemination of the fungus Fusarium oxysporum f. sp. fragariae.
 To identify the presence of the pathogen, by applying molecular techniques to the Fusarium strains isolated from strawberry crops.
 Methods: Nine two diseased strawberry plants and 92 asymptomatic plants were analyzed. From these samples, 13 fungi with the characteristics of the Fusarium genus were identified. The isolates were analyzed at the molecular level, by PCR (Polymerase chain reaction) amplifying the ITS regions of the rDNA and the EF-1α region.
 Results: The PCR product was sequenced to elucidate the phylogenetic relationships between the isolates, identifying 12 strains as F. oxysporum f. sp. fragariae. These results confirmed the presence of the fungus in the strawberry crops analyzed, representing a contribution to the search for control alternatives to avoid the spread of the pathogen.
 Conclusion: The PCR product was sequenced to elucidate the phylogenetic relationships between the isolates, identifying 12 strains as F. oxysporum f. sp. fragariae.

Untargeted Metabolomics Based on GC-MS and Chemometrics: A New Tool for the Early Diagnosis of Strawberry Anthracnose Caused by Colletotrichum theobromicola.

To prevent the spread of anthracnose in strawberry plants and characterize the metabolic changes occurring during plant-pathogen interactions, we developed a method for the early diagnosis of disease based on an analysis of the metabolome by gas chromatography-mass spectrometry. An examination of the metabolic profile revealed 189 and 202 total ion chromatogram peaks for the control and inoculated plants, respectively. A partial least squares discriminant analysis (PLS-DA) model was conducted for the reliable and accurate discrimination between healthy and diseased strawberry plants, even in the absence of disease symptoms (e.g., early stages of infection). ANOVA (analysis of variance) and orthogonal partial least squares analysis (OPLS) identified 20 metabolites as tentative biomarkers of Colletotrichum theobromicola infection (e.g., citric acid, d-xylose, erythrose, galactose, gallic acid, malic acid, methyl α-galactopyranoside, phosphate, and shikimic acid). At least some of these potential biomarkers may be applicable for the early diagnosis of anthracnose in strawberry plants. Moreover, these metabolites may be useful for characterizing pathogen infections and plant defense responses. This study confirms the utility of metabolomics research for developing diagnostic tools and clarifying the mechanism underlying plant-pathogen interactions. Furthermore, the data presented herein may be relevant for developing new methods for preventing anthracnose in strawberry seedlings cultivated under field conditions.

Metabolic Profiling to Identify the Latent Infection of Strawberry by Botrytis cinerea.

In plant-pathogen interaction systems, plant metabolism is usually agitated in the early stages of infection and much before visible symptoms appear. To identify the latent infection of strawberry by Botrytis cinerea by metabolome profiling, a metabolomics method based on gas chromatography and mass spectrometry was applied to identify the affected metabolites and discriminate diseased plants from healthy ones. An orthogonal partial least squares (OPLS) score plot showed that the metabolic profiling well separated B. cinerea-infected strawberry plants at 2, 5, and 7 days after infection from non-infected healthy plants. Combined analysis of variance (ANOVA) and OPLS analysis revealed candidate biomarkers of plant resistance and of infection and expansion of the pathogen in the plants. Among them, hexadecanoic acid, octadecanoic acid, sucrose, β-lyxopyranose, melibiose, and 1,1,4a-Trimethyl-5,6-dimethylenedecahydronaphthalene were closely related to the early stage of disease development when symptoms were not visible. A discrimination method that could distinguish Botrytis gray mold diseased strawberry plants from healthy ones was established based on the partial least squares discriminant analysis (PLS-DA) model with a correct recognition accuracy of 100%. This research offers a good application of metabolome profiling for early diagnosis of plant disease and interaction mechanism exploration.

A TaqMan real-time polymerase chain reaction assay for accurate detection and quantification of Fusarium solani in strawberry plants and soil

Abstract A new TaqMan real-time polymerase chain reaction (qPCR) was developed to accurately detect and quantify the soil-borne fungus Fusarium solani, pathogen to strawberry, in plant and soil samples. The assay was designed using sequences from the translation elongation factor 1 alpha (EF-1α) gene. The assay is highly specific: it specifically detected F. solani isolates when tested against 95 fungal isolates from 27 different species. The detection limit of the assay was 50 fg for F. solani genomic DNA and 102 conidia/g soil. A significant correlation (P = 0.0002) was observed between the amount of genomic DNA of F. solani detected by qPCR and the number of fungal propagules present in artificially inoculated soils. The effectiveness of the assay was validated by comparing it to traditional methods for the detection of F. solani in diseased strawberry plants and pre-planting soils from strawberry fields. A substantial and moderate correlation was found between the qPCR-based and traditional detection methods in diseased plants and soils. The amount of F. solani DNA estimated in roots and crowns of symptomatic strawberry plants ranged from 16 to 190 pg/mg fresh tissue. Inoculum densities in pre-planting soils varied between 3.1 × 102 and 1.3 × 105 colony-forming units per gram (CFU/g) of soil. Effectiveness was also evaluated by assessing the ability of the assay to detect decreasing levels of F. solani populations during biosolarization treatment. Taken together, this novel qPCR assay represents a useful tool for rapid assessment of pre-planting soils and nursery plants to prevent F. solani infection and production losses.

New foci of strawberry Fusarium wilt in Huelva (Spain) and susceptibility of the most commonly used cultivars

Abstract Strawberry Fusarium wilt is becoming an important disease after methyl bromide banning. In southwestern Spain, this disease was previously detected in a soilless culture system and several years later only one isolation was reported from plant cultivated in soil. This study reports the appearance of new foci of strawberry Fusarium wilt in plants cultivated on soils and evaluates the susceptibility of nine of the most commonly grown cultivars in this zone. Fungal isolates were obtained from diseased strawberry plants in the nineteen new foci showing an increase of incidence and geographic range of this disease. These isolates were morphologically and molecularly characterized as Fusarium oxysporum . Fofra amplicons were not obtained and only eight isolates amplified for Fusarium transcription factor 1 . Pathogenicity of six selected isolates as well as a Fusarium oxysporum f. sp. fragariae culture from Japan were tested using by inoculating cultivar ‘Splendor’. All the Spanish isolates were found to be pathogenic. As a follow-up study, nine strawberry cultivars were inoculated with the Japanese isolate and a mixture of three Spanish isolates. The least susceptible cultivars to the Spanish isolates were ‘Rociera FNM’, ‘San Andreas’, ‘Camarosa’, ‘Sabrina’ and ‘Sabrosa Candonga’. Cultivar susceptibility was different with respect to Japanese isolate.

The Population of Fusarium oxysporum f. sp. fragariae, Cause of Fusarium Wilt of Strawberry, in California.

The objectives of this study were to investigate the structure of the population of Fusarium oxysporum f. sp. fragariae in California and to evaluate methods for its detection. Fifty-nine isolates of F. oxysporum f. sp. fragariae were obtained from diseased strawberry plants and their identity was confirmed by pathogenicity testing. The full nuclear ribosomal intergenic spacer (IGS) and elongation factor 1-α gene (EF-1α) were amplified by polymerase chain reaction (PCR) and sequenced to elucidate phylogenetic relationships among isolates. IGS and EF-1α sequences revealed three main lineages, which corresponded to three somatic compatibility groups. Primers designed to detect F. oxysporum f. sp. fragariae in Japan amplified a 239-bp product from 55 of 59 California isolates of F. oxysporum f. sp. fragariae and from no nonpathogenic isolates of F. oxysporum. The sequence of this PCR product was identical to the sequence obtained from F. oxysporum f. sp. fragariae isolates in Japan. Intensive sampling at two locations in California showed results of tests based on PCR and somatic compatibility to be in agreement for 97% (257 of 264) of isolates tested. Our findings revealed considerable diversity in the California population of F. oxysporum f. sp. fragariae, and indications that horizontal gene transfer may have occurred.

Wide variation in virulence and genetic diversity of binucleate Rhizoctonia isolates associated with root rot of strawberry in Western Australia.

Strawberry (Fragaria×ananassa) is one of the most important berry crops in the world. Root rot of strawberry caused by Rhizoctonia spp. is a serious threat to commercial strawberry production worldwide. However, there is no information on the genetic diversity and phylogenetic status of Rhizoctonia spp. associated with root rot of strawberry in Australia. To address this, a total of 96 Rhizoctonia spp. isolates recovered from diseased strawberry plants in Western Australia were characterized for their nuclear condition, virulence, genetic diversity and phylogenetic status. All the isolates were found to be binucleate Rhizoctonia (BNR). Sixty-five of the 96 BNR isolates were pathogenic on strawberry, but with wide variation in virulence, with 25 isolates having high virulence. Sequence analysis of the internal transcribed spacers of the ribosomal DNA separated the 65 pathogenic BNR isolates into six distinct clades. The sequence analysis also separated reference BNR isolates from strawberry or other crops across the world into clades that correspond to their respective anastomosis group (AG). Some of the pathogenic BNR isolates from this study were embedded in the clades for AG-A, AG-K and AG-I, while other isolates formed clades that were sister to the clades specific for AG-G, AG-B, AG-I and AG-C. There was no significant association between genetic diversity and virulence of these BNR isolates. This study demonstrates that pathogenic BNR isolates associated with root rot of strawberry in Western Australia have wide genetic diversity, and highlights new genetic groups not previously found to be associated with root rot of strawberry in the world (e.g., AG-B) or in Australia (e.g., AG-G). The wide variation in virulence and genetic diversity identified in this study will be of high value for strawberry breeding programs in selecting, developing and deploying new cultivars with resistance to these multi-genetic groups of BNR.

First Report of Anthracnose on Fruits of Duchesnea indica Caused by Colletotrichum acutatum in Northwestern Argentina.

Duchesnea indica (Andrews) Focke, a cosmopolitan wild species related to the cultivated strawberry that is widely distributed in northwestern Argentina, grows in close proximity to strawberry crops and has proven to be almost immune to Colletotrichum spp. isolated from diseased strawberry plants (1), hence it has never been considered a phytopathological risk. During a field survey of "La Heladera" (27°01'45″S, 65°39'20″W), Tafí del Valle (Tucumán, Argentina) from November 2009 to November 2010, a genotype of D. indica showing fruits with dark brown, necrotic, irregular, circular lesions of 5 to 20 mm in diameter were collected. Setose acervuli were observed on the center of the fruit lesions. Pathogens were obtained from 10 diseased fruit collected at random, and four fungal isolates were isolated per fruit on potato dextrose agar (PDA). To reduce the number of samples for evaluation, two isolates per fruit that were exhibiting stable but distinctive morphological features were chosen to continue the studies. Isolates were characterized by morphological, molecular, and phytopathological criteria. After 10 days of incubation on PDA medium at 28°C with continuous white light, colonies exhibited a gray, aerial mycelium, whereas the reverse of the colony is a pale maroon with a radial, pale salmon color. Masses of salmon-colored conidia formed in the center of the colonies. Conidia were hyaline, one celled, fusiform, tapered to a point at both ends, and measured 14.8 to 17.3 × 4.5 to 7.4 μm (n = 100). Setae were scarce and sclerotia were absent. All morphological characteristics that were observed indicated that the isolates were C. acutatum (3). To fulfill Koch's postulates and verify the pathogenicity on commercial varieties of strawberry, six healthy plants of D. indica and Fragaria × ananassa cv. Camarosa with mature fruits were used to test each isolate. Four plants were spray inoculated with conidial suspensions of the virulent isolates (1.5 × 106 conidia/ml) and two with sterile distilled water as controls. Both treatments were maintained under white light (2,000 lux, 12 h per day) at 28°C and 70% relative humidity. Nine days after the inoculation, dark brown lesions and salmon-colored masses of conidia were observed only in inoculated fruits of both genotypes. The fungus isolated from diseased fruits and the conidia that were produced were identical to the isolates used to inoculate the plants. To confirm pathogen identity, PCR amplification with the species-specific pair of primer CaInt2/ITS4 (4) were carried out using fungal total DNA from the original isolates and isolates obtained from inoculated fruits. An amplification product of approximately 490 bp, which is specific for C. acutatum, was observed in all DNA samples (4). Although C. acutatum has already been reported in Fragaria × ananassa in Argentina (2), to our knowledge, this is the first report of C. acutaum causing anthracnose in D. indica species. This result is relevant since this species grows close to strawberry fields and can be an alternative host and potential vector of the anthracnose disease agent.

First Report of Curvularia lunata Causing Root Rot of Strawberry in India.

Diseased strawberry (Fragaria × ananassa Dutch) plants were observed during semimonthly surveys in the Jammu District of Jammu and Kashmir State, India from November 2007 to May 2008. Symptoms included leaf wilt, necrotic roots, and plant death. Small pieces of symptomatic roots were cut from the junction of diseased and healthy tissue, surface sterilized in 0.1% mercuric chloride solution for 1 min, washed in three changes of sterile distilled water, and transferred to potato dextrose agar plates. Cultures were maintained in an incubator at 25 ± 1°C. Dark brown fungal colonies developed in a few days. Mycelium was septate, dark brown-to-blackish brown, and branched. Conidiophores were dark brown, unbranched and septate. Conidia were boat shaped or curved, dark brown, and four-celled with the two central cells larger than the terminal cells. Apical cells were light brown, rounded at the tips, slightly constricted at the base, and 19 to 30 × 9 to 14 μm. The pathogen was identified by the Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India (ITCC Accession No. 6680-07) as Curvularia lunata (Wakkar) Boevijn. To conduct pathogenicity tests, 10 healthy runners of strawberry cv. Chandler were planted separately in sterilized pot mix containing loam soil, sand, and peat moss (1:1:1 vol/vol) and allowed to root for a month. Soil near the root zone was then removed to access roots that were gently injured by pricking with a sterilized needle. The soil was replaced after 20 ml of a conidial suspension of 106 conidia/ml of one of the isolates was applied to the roots. Ten healthy strawberry plants grown in noninoculated soil served as controls. After inoculation, the plants were regularly watered. Symptoms similar to those observed in the field developed on all inoculated plants. Roots of the diseased plants turned brown and finally black. Noninoculated plants remained healthy. The pathogen was reisolated from roots of the diseased plants. Curvularia spp. are ubiquitous and are typically considered to be weak pathogens or saprophytes; however they have been reported as minor pathogens of several plants (1,2). Root rot of strawberry caused by Curvularia spp. has been reported (3), but to our knowledge, this is the first report of C. lunata (Wakkar) Boevijn causing root rot of strawberry in India.

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first‐round PCR products was used as template with two P. cactorum‐specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340‐bp fragment and a 480‐bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431‐bp fragment). Validation tests using culture‐based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.

First Report of Strawberry vein banding virus on Strawberry in Italy.

In the summers of 2007 and 2008, diseased strawberry plants (Fragaria x ananassa Duch.) were observed in production fields in Ferrara and Forlì-Cesena districts (Emilia-Romagna Region) in northern Italy. Plants exhibited poor growth, leaf chlorosis, decline, and reduced fruit production. Older leaves sometimes displayed a premature purplish discoloration, while the younger leaves appeared chlorotic and were reduced in size with a marked yellow edge. Symptom severity was dependent on the cultivar and growing conditions. Sixty-one leaf samples were collected from diseased plants from production fields and cultivar collections. Each sample was tested by grafting on Fragaria vesca (clone 'UC4 and UC5') and F. virginiana (clone 'UC10' and 'UC11'). Forty-five days after indexing different symptoms, characteristic of viral diseases, appeared on indicator plants. In particular, 24 samples showed Strawberry vein banding virus (SVBV)-like symptoms with chlorotic streaks along and on both sides of the main leaf veins of UC5, UC10, and UC11 indicator plants. Molecular methods were also used to better investigate the causal agent. Nucleic acids were extracted from young leaves of field and indicator plants by the cetyltrimethylammoniumbromide method (1). PCR analyses were performed with primer pair SVBVdeta/SVBVdetb as previously described to specifically amplify a product of 423 bp (2). SVBV was detected on all symptomatic indicator plants and corresponding field samples as well as on the positive control sample (supplied by J. D. Postman, National Clonal Germplasm Repository, Corvallis, OR and I. E. Tzanetakis, Oregon State University, Corvallis). No amplicons were detected from nucleic acids extracted from symptomless strawberry plants. PCR products, amplified from four Italian SVBV isolates, were cloned and sequenced and represent part of ORF IV of the SVBV genome that codes for the coat protein (CP). Italian SVBV isolates were more similar to the U.S. isolates than to the Chinese isolates (Genbank Accession Nos. AY862389, AY955374, X97304, AY605662, AY605663, and AY605664), showing 93 and 86% nt sequence identity, respectively. Strawberry vein banding disease has been reported previously in Italy in 1986 (3), but to our knowledge, this is the first finding of SVBV on strawberry field plants in Italy. SVBV was listed as a quarantine pest by the European and Mediterranean Plant Protection Organization (OEPP/EPPO) in 1978, but its spread has been increasing within European countries. Further studies should be done to ensure that strawberry propagating material is free of known viruses including SVBV.

Crown Rot of Strawberry Caused by Macrophomina phaseolina in California.

In 2006 and 2007, severely diseased strawberry (Fragaria × ananassa) plants were observed in five commercial fields in southern California (Orange County). Disease generally occurred in discrete patches. Within such patches, disease incidence ranged from 10 to 75%. Symptoms consisted of wilting of foliage, drying and death of older leaves, plant stunting, and eventual collapse and death of plants. When plant crowns were dissected, internal vascular and cortex tissues were dark brown to orange brown. Fruiting bodies or other fungal structures were not observed. A fungus was consistently isolated from symptomatic crown tissue that had been surface sterilized and placed on acidified corn meal agar (LA-CMA). All isolates produced numerous, dark, irregularly shaped sclerotia that were 67 to 170 μm long and 44 to 133 μm wide. When isolates were grown on 1.5% water agar with dried and sterilized wheat straw, dark, ostiolate pycnidia and hyaline, single-celled, cylindrical conidia were produced. On the basis of these characters, all isolates were identified as Macrophomina phaseolina (1). The symptomatic plants tested negative for Colletotrichum spp., Phytophthora spp., Verticillium dahliae, and other pathogens. Inoculum for pathogenicity tests was produced by growing six isolates on CMA on which sterilized wood toothpicks were placed on the agar surface. After 1 week, toothpicks were removed and inserted 4 to 5 mm deep into the basal crown tissue of potted strawberry plants (cv. Camarosa) grown in soilless, peatmoss-based rooting medium. Ten plants were inoculated per isolate and one toothpick was inserted per plant. Ten control strawberry plants were treated by inserting one sterile toothpick into each crown. All plants were then grown in a shadehouse. After 2 weeks, all inoculated plants began to show wilting and decline of foliage. By 4 weeks, all inoculated plants had collapsed. Internal crown tissue was discolored and similar in appearance to the original field plants. M. phaseolina was isolated from all inoculated plants. Control plants did not exhibit any disease symptoms, and crown tissue was symptomless. The test was repeated and the results were similar. While M. phaseolina has been periodically associated with strawberry in California (3), to my knowledge, this is the first report of charcoal rot disease on commercial strawberry in California. Charcoal rot of strawberry has been reported in Egypt, France, India, Israel, and the United States (Florida and Illinois) (2,4). Similar to previous reports (2,4), many of the affected California fields were not preplant fumigated with methyl bromide + chloropicrin fumigants, and it is possible that under these changing production practices this pathogen may increase in importance in California.

First Report of a γ 3-Proteobacterium Associated with Diseased Strawberries in Italy.

During the fall seasons of 2005 and 2006, diseased strawberry plants (Fragaria × ananassa Duch.) were observed in nurseries and production fields in Ferrara, Forli-Cesena, and Ravenna provinces (Emilia-Romagna region, northern Italy). Symptoms consisted of a conspicuous plant stunting with a poor root system. Older leaves rolled upward and displayed a marked premature purplish discoloration, while young leaves were cupped, chlorotic, generally reduced in size, and had shortened petioles. This strawberry disorder was similar to "marginal chlorosis", an infectious disease occurring in France that can be induced by two different phloem-limited uncultured bacteria: the γ 3-proteobacterium 'Candidatus Phlomobacter fragariae' and the stolbur phytoplasma (16SrXII-A). In strawberry production fields, 'Ca. P. fragariae' is reported as being the prevalent agent of this disease (1). Sixty-seven diseased plants were collected from production fields and nurseries for testing for 'Ca. P. fragariae'. Leaf samples were analyzed by 4',6-diamidine-2-phenylindole staining and PCR. Forty samples showed fluorescent DNA in the phloem, whereas no fluorescence was observed in symptomless strawberries. When tested by PCR with primers Fra4/Fra5, which amplify a 550-bp fragment of the 16S rDNA region of 'Ca. P. fragariae' (1), 13 of 36 strawberries from production fields and 1 of 31 nursery plants gave a positive reaction. On the other hand, 21 samples from nurseries and 5 from production fields tested positive for stolbur phytoplasma (3). No amplification was obtained with DNA from symptomless or healthy strawberry plants. Sequencing Fra4/Fra5 amplicons from three samples (GenBank Accession Nos. DQ362916-DQ362918) showed a 98.1 to 98.6% and a 98.3 to 98.8% identity with the published sequences of the French isolate "LG2001" (GenBank Accession No. AM110766) and the Japanese isolate J-B (GenBank Accession No. AB246669) of 'Ca. P. fragariae', respectively. Higher homology (99.2 to 99.8%) was found with another bacterium-like organism (BLO) of the γ 3-proteobacteria subgroup (GenBank Accession No. AY057392) associated with the syndrome "basses richesses" of sugar beet (SBR). Furthermore, PCR assays performed with primers Pfr1/Pfr4, specific for spoT gene of 'Ca. P. fragariae', did not show any amplification with DNA from the 14 diseased strawberry plants tested. This is in agreement with the SBR BLO identification (2). To better characterize the Italian isolates, the full-length 16S rDNA gene was analyzed with primers fd1/Fra4 and Fra5/rp1, which amplify the 5' and 3' region of 16S rDNA gene of the proteobacteria, respectively (2). PCR products from eight isolates were sequenced, and the 16S rDNA sequences obtained (GenBank Accession Nos. DQ538372-DQ538379) showed a 96.4 to 97.3% identity with the known 'Ca. P. fragariae' isolates, while a higher homology (99.4 to 99.9%) was again found with the SBR BLO. To our knowledge, this is the first report of a γ 3-proteobacterium affecting strawberry in Italy. In the genome region analyzed, our isolates are more similar to the SBR BLO than to 'Ca. P. fragariae'. Further work is in progress to investigate incidence, geographical distribution, epidemiology, and host range of this pathogen in Italy.

Characterization of Fusarium oxysporum f. sp. fragariae Based on Vegetative Compatibility Group, Random Amplified Polymorphic DNA and Pathogenicity

Twenty-two isolates of Fusarium oxysporum f. sp. fragariae were obtained from diseased strawberry plants and their characteristics were investigated by vegetative compatibility group (VCG), random amplified polymorphic DNA (RAPD), and pathogenicity. Three major VCGs (A, B, and C) and one incompatible group were identified by nitrate reductase complementation test. The virulence pattern of the 22 isolates was studied in relation to four cultivars including Dochiodome, Red-pearl, Maehyang and Akihime. RAPD markers were used to determine genetic relationship, and created three major clusters among the 22 isolates of F. oxysporum f. sp. fragariae. Isolates belong to VCG-C were strongly pathogenic, and relatively high correlation was existed among VCG and RAPD, and virulence. In addition, VCG and RAPD pattern between pathogenic and non-pathogenic isolates were distinctly different.

First Report of Stolbur Phytoplasma (16SrXII-A) on Strawberry in Northern Italy.

Strawberry (Fragaria × ananassa Duch.) is one of the most important small-fruit crops in northern Italy. During the autumn of 2003, in nurseries located in Ravenna Province (Emilia-Romagna Region), a disease characterized by pronounced stunting and a very poor root system was observed in plants of the cv. Tethis. Older leaves of diseased plants were rolled upward and displayed a marked premature purple discoloration; new leaves showed size reduction, shortened petioles, chlorosis, and were generally cupped. Some of these plants were potted and kept in greenhouse conditions; the following spring, they exhibited typical floral abnormalities as virescent and phylloid petals. Flowers were fully or partly sterile, producing small and deformed fruits; new foliage was dwarfed, asymmetrical, and pale green with chlorotic margins. Later, the affected plants expressed a quick decline consisting of growth cessation, bronzing of mature leaves, wilting, and death. This strawberry yellows-type disease was suggestive of a phytoplasmal infection. Symptoms were identical to "marginal chlorosis", a stolbur-associated disease occurring in France (4). To acquire more information, field inspections were extended to the 2004 and 2005 seasons. Additional cultivars (Alba, Aromas, Camarosa, Gemma, Maya, NF 20, Queen Elisa, Roxana, and Selva) affected by a similar disorder were identified in strawberry nurseries and production fields from different sites of Ravenna and Forlì-Cesena provinces. Total DNA extracted from collected plants was tested using nested polymerase chain reaction (nPCR) performed with universal phytoplasma primers P1/P7, followed by phytoplasma-specific primer pair R16F2/R2 or group 16SrI and 16SrXII-specific primer pair R16(I)F1/R1 (1,2). Results from nPCR revealed that 21 of 23 diseased nursery plants were infected by a phytoplasma. On the contrary, no positive reaction was obtained with diseased strawberry plants collected from production fields. Subsequent restriction fragment length polymorphism analysis of the nPCR-amplified product R16(I)F1/R1 with enzyme MseI indicated that all diseased plants contained the same phytoplasma belonging to the phytoplasma subgroup 16SrXII-A. Subsequently, these results were confirmed by nPCR using group 16SrXII specific primer pair fSTOL/rSTOL (1). The fragments amplified from three samples were sequenced (GenBank Accession Nos. DQ350615-DQ350617) and showed 99.6 to 99.8% nucleotide sequence identity with a grapevine stolbur isolate (GenBank Accession No. AJ964960). In addition, all samples were assayed using nPCR with primer pair fTuf1/rTuf1 and primers fTufAy/rTufAy, specific for groups 16SrI and 16SrXII (1). Results showed the presence of an expected 945-bp product from infected samples. Sequencing of five amplicons (GenBank Accession Nos. DQ418456-DQ418460) shared 99.4 to 99.9% nucleotide sequence homology with a periwinkle stolbur isolate (GenBank Accession No. L46370). Before now, stolbur phytoplasma has been found to be associated with a strawberry plant showing phyllody symptoms in southern Italy (3). Our report is a wider demonstration of this pathogen infecting strawberry in major cultivations areas of northern Italy.