Calendula officinalis: A New Natural Host of Pseudomonas viridiflava in Italy.

In November 2010, small necrotic spots surrounded by chlorotic halos, which sometimes enlarged and coalesced to form large dead areas, were observed on leaves of marigold (Calendula officinalis L.) plants grown in the Medieval Garden at the Agricultural Faculty of Perugia (central Italy). Cream-colored bacterial colonies were consistently isolated on nutrient agar (NA) from the diseased leaf tissues. Four representative selected strains, which were gram negative, fluorescent on King's medium B, and had oxidative but not fermentative metabolism, were subjected to a pathogenicity test by inoculating 1-month-old marigold plants. To prepare the inoculum, the bacterial strains were grown on NA at 27°C for 24 h, suspended in sterile deionized water, and adjusted to 1 × 106 CFU/ml. Sterile water was used for control plants. Marigold leaves were infiltrated with a glass atomizer at high pressure, and plants were kept in a growth chamber at 22 to 24°C, 70 µE·m-2·s-1 illumination and 12-h light period, and 80% relative humidity. Small, water-soaked necrotic spots were observed 10 days after inoculation, and the bacterium with the same cultural features of the original strains was reisolated from inoculated plants. For bacterial identification, the four original strains and two reisolates were subjected to LOPAT tests. They were levan negative, oxidase negative, potato rot positive, arginine dihydrolase negative, and tobacco hypersensitive response positive. These results were similar to those obtained with the type strain LMG 2352T of Pseudomonas viridiflava (Burkholder) Dowson. When 16S rDNA was amplified with the universal primers, P0 (6-27f Escherichia coli) and P6 (1515-1495r E. coli), and digested with the endonucleases, SacI and HinfI as previously reported (2), an identical restriction profile was obtained for marigold strains and reisolates and P. viridiflava strains, LMG 2352T, LMG 2353, LMG 5397, and NCPPB 1382. A completely different profile was obtained for P. syringae pv. syringae LMG 1247T. The 16S rDNA (1,364 bp) and the gyrB (570 bp) sequences of two selected marigold strains (GenBank Accession Nos. JN406504 and JN406505; JN406506 and JN406507), amplified by using universal and previously reported PCR primers (3), respectively, shared 100% sequence identity with P. viridiflava (GenBank Accession Nos. HM190229 and AY606763) for 16S rDNA and gyrB gene, respectively. On the basis of biochemical, physiological, molecular, and pathogenicity tests, it was concluded that the bacteria isolated from marigold leaves are P. viridiflava. To our knowledge, this is the first report of C. officinalis as a natural host of P. viridiflava. The plant was previously reported as a host of the bacterium by artificial inoculation (1). References: (1) J. F. Bradbury. Guide to Plant Pathogenic Bacteria. CAB International, Egham, UK, 1986. (2) A. J. González et al. Appl. Environ. Microbiol. 69:2936, 2003. (3) E. M. Goss et al. Genetics 169:21, 2005.

PCR-based assay for the detection of Xanthomonas euvesicatoria causing pepper and tomato bacterial spot.

AIMS: To develop a PCR-based assay for Xanthomonas euvesicatoria detection in culture and in planta. METHODS AND RESULTS: A fragment of 1600 bp specific for X. euvesicatoria was found by repetitive extragenic palindromic sequence-PCR. Among the primers designed on the basis of the partially sequenced fragment, the primers Xeu2.4 and Xeu2.5 direct amplification of the expected product (208 bp) for all the X. euvesicatoria strains and not for other related and unrelated phytopathogenic bacteria or saprophytic bacteria isolated from pepper and tomato phyllosphere. The assay permits the detection of X. euvesicatoria in pure culture, with a limit of detection of two bacterial cells and 1 pg of DNA per PCR, and in extracts obtained from asymptomatic inoculated tomato and pepper plants. CONCLUSIONS: Primers Xeu2.4 and Xeu2.5 provide a specific, sensitive and rapid assay for the detection of X. euvesicatoria in culture and in pepper and tomato plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Because X. euvesicatoria is a quarantine organism in the European Union, and it is subjected to stringent international phytosanitary measures, this highly sensitivity PCR-based assay is suitable for its detection in pepper and tomato plant materials to avoid the introduction and spread of the bacterium.

First Report of Leaf Necrosis Caused by Pseudomonas viridiflava on Melon Seedlings in Italy.

In April 2001, necrotic lesions surrounded by thin, water-soaked halos were observed on cotyledons of 12-day-old melon seedlings (Cucumis melo var. reticulatus, cv. Baggio, Calipso, and Proteo) grown in plant beds in an unheated greenhouse located in the Province of Perugia (central Italy). The incidence of the disease was approximately 10%, and the economic impact was limited as seedlings recovered from the disease. Cream-colored, mucoid, bacterial colonies were consistently isolated on nutrient agar from the diseased leaf tissues. Two representative strains selected for identification were gram negative, fluorescent on King's medium B, and had oxidative but not fermentative metabolism. They were levan negative, oxidase negative, potato rot positive, arginine dihydrolase negative, and tobacco hypersensitive response positive in LOPAT tests. These isolates showed pectolytic activity at pH 8 but not at pH 4 and utilized L-arabinose, D(-)-tartrate and L-lactate. They did not utilize sucrose, L(+)-tartrate, or trigonelline, and did not produce acid from sucrose after 21 days of incubation. These results were similar to those obtained with the type strain LMG 2352T of Pseudomonas viridiflava (Burkholder) Dowson. Although suitable for strain characterization, we found that repetitive sequence-based polymerase chain reaction (rep-PCR) conducted with primer BOXA1R was not appropriate for identifying P. viridiflava. In fact, each P. viridiflava strain tested, (LMG 2352T, LMG 2353, LMG 5397, and NCPPB 1382) generated unique fingerprints, which differed from the two melon strains. Pathogenicity tests were carried out with 3-week-old melon (cv. Baggio), cucumber (cv. Lungo verde degli ortolani), and zucchini (cv. Consul) plants (three plants for each species and isolate). To prepare the inoculum, the two bacterial strains were grown on nutrient agar for 24 h at 27°C, suspended in sterile deionized water, and adjusted to 1 × 106 CFU ml-1. Young leaves of melon, cucumber, and zucchini plants infiltrated with bacterial suspensions by a glass atomizer at high pressure developed small, chlorotic spots with necrotic centers surrounded by water-soaked halos 5 to 7 days after inoculation. The most severe symptoms were observed on melon plants. The two strains also induced severe symptoms (chlorosis and water soaking) on Arabidopsis thaliana (ecotype Columbia-0) 3 to 4 days after inoculation. No symptoms were observed in control plants. The bacterium was readily recovered from inoculated plants, and their rep-PCR fingerprints were identical to the strains used for inoculation. On the basis of biochemical, physiological, nutritional, and pathogenicity tests, it was concluded that the bacteria isolated from symptomatic melon seedlings were P. viridiflava. To our knowledge, this is the first report of P. viridiflava attacks on melon plants in Italy. The disease was previously recorded in Turkey (1) and Greece (2). References: (1) A. Aysan et al. Plant Pathol. 52:800, 2003. (2) D. E. Goumans and A. K. Chatzaki. Eur. J. Plant Pathol. 104:181, 1998.

Occurrence of Anthracnose Caused by Colletotrichum malvarum on Althaea officinalis in Italy.

The cultivation of medicinal plants is increasing in some areas of central Italy where the climate is suitable for organic farming and the production of high-quality plant products. During April and May 2003, plants of Althaea officinalis L. at the seedling stage (two-to-four true leaves) maintained in unheated greenhouses before their transplantation to open fields showed an unusual foliar disease. Necrotic leaf spots of variable shape and size were followed by a rapid wilting of leaves that frequently resulted in a blight of the young plants. Small leaf pieces showing symptoms were sampled, surface treated in 0.1% HgCl2 for 30 s, rinsed twice in sterile water, placed on potato dextrose agar (PDA) (pH 5.5) in petri dishes, and incubated for 7 days at 25 ± 2°C. Colletotrichum malvarum (Braun & Casp.) Southworth (1,2) was consistently recovered from affected tissues. The fungus produced dark colonies with whitish aerial mycelium and acervuli containing hyaline, cylindrical conidia (14 to 25 × 3 to 6 µm) on PDA. The pathogenicity of four fungal isolates was tested by inoculating two, true leaves of 10 plants (A. officinalis) with a conidial suspension (5 × 105 conidia ml-1) from a 10-day-old culture. Plants sprayed with water served as controls. All seedlings were placed in a greenhouse at 24± 2°C under natural light conditions and covered with plastic bags for the first 24 h. Each pathogenicity test was repeated one time. After 5 to 7 days, the inoculated seedlings showed small necrotic leaf spots identical to those observed under natural conditions. Affected leaf areas rapidly enlarged and within a few days, the young plants wilted. No symptoms appeared on the noninoculated controls. C. malvarum was consistently reisolated from the symptomatic test seedlings, whereas the fungus was never isolated from control plants. Standard seed health methods (agar plate and blotter) carried out on samples from the same seed lots used for the unheated greenhouse trials were negative for the presence of the pathogen. The occurrence of anthracnose may be attributed to windborne conidia of C. malvarum coming from infected wild malvaceae species and cultivated hosts grown in open fields in the neighborhood of seedling greenhouses. To our knowledge, this is the first report of C. malvarum on A. officinalis in Italy. References: (1) W. Brandenburger. Page 386 in: Parasitische pilze an gefäbpflanzen in Europa. Fisher Verlag, Stuttgart, Germany, 1985. (2) B. C. Sutton. The genus Glomerella and its anamoroph Colletotrichum. Pages 1-26 in: Colletotrichum, Biology, Pathology and Control. J. A. Bailey and M. J. Jeger eds. CAB International, Wallingford, U.K., 1992.

Occurrence of Pseudoperonospora cubensis Pathotype 5 on Squash in Italy.

During the period of May to August 2002, typical symptoms of downy mildew were observed on squash (Cucurbita pepo L.) in both tunnel and open field cultivation in central Italy (Latium and Umbria). The disease spread rapidly and because the control measures used were not effective, growers suffered severe yield losses. Infected plants showed yellow spots on the upper leaf surface. Based on morphological features observed at ×10 to ×40 magnification, the pathogen was identified as Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev. To verify the pathogenicity of the fungus, a sporangial suspension (1 × 104 sporangia per ml) was sprayed on leaves of squash plants with two expanded leaves, which were held in dark moist chambers at 20 ± 2°C for 48 h. Control plants were sprayed with sterile water. Inoculated and control plants were kept in a growth chamber at 24 ± 2°C with 14/10 h day/night cycles. Chlorotic spots and sporulation were observed on inoculated plants. The morphological features of the fungus obtained from the inoculated plants were identical to those from the original diseased plant. Previous observations of downy mildew in Italy suggested that squash was not susceptible (1). The physiological specialization among isolates of P. cubensis based on the compatibility of different cucurbit hosts (2) was not tested previously in Italy. To determine the pathotype of four fungal isolates obtained from squash cultivated in different localities, 10 plants per species of the cucurbit species (2) were inoculated with each isolate using the same procedure described for the pathogenicity test. Disease symptoms were detected on all inoculated hosts, including squash, suggesting that all the fungal isolates obtained from squash are pathotype 5. Only pathotype 5 is a common causal agent of downy mildew of squash and other cucurbit hosts. During the period of our observations, climatic conditions were unusually wet because frequent storms occurred during the summer providing favorable environmental conditions for the development of secondary spread. Today, it appears there are no commercially acceptable cultivars of squash resistant to downy mildew available to growers in Italy. References: (1) F. Ciccarese et al. Phytopathol. Mediterr. 29:14, 1990. (2) C. E. Thomas et al. Phytopathology 77:1621, 1987.

Occurrence of Black Dot of Potato Caused by Colletotrichum coccodes in Central Italy.

Between 1997 and 2000, black dot of potato (Solanum tuberosum L.), caused by the polyphagous soilborne fungus Colletotrichum coccodes (Wallr.) Hughes, was observed each summer in fields located in Umbria (central Italy). Disease incidence ranged from 50 to 100%, and early potato cultivars were generally more susceptible than late-maturing ones. Disease symptoms were first observed during August as a yellowing and wilting of foliage in the tops of plants, followed by rotting of the roots and stems, which led to the premature death of 50 to 70% of plants. Setose1 sclerotia (300 to 500 mm in diameter) and acervuli of the fungus were found on roots and stems of infected plants. Acervuli produced hyaline, aseptate, cylindrical conidia (16 to 22 × 2.5 to 4.5 µm) formed on unicellular cylindrical phialidic conidiophores. The fungus was isolated from diseased stems and roots on potato dextrose agar (PDA) at pH 6.5. Pathogenicity of the fungus was confirmed by fulfilling Koch's postulates using 3- to 4-week-old potato plants of a local cultivar. A superficial 5-mm vertical cut was made with a scalpel into the base of potato stems (2 cm beneath the soil surface), and 5-mm-diameter plugs of PDA alone (control plants) or PDA plus fungal growth were placed over the cuts. The wounds were sealed with wet cotton swabs that were held in place with Parafilm. Symptoms that resembled those in the field were observed on inoculated plants 6 to 8 weeks postinoculation. Symptoms did not appear on the control plants. The same fungus was reisolated from the diseased plants. Based on morphological characteristics of sclerotia, acervuli, and conidia, as well as pathogenicity tests, the fungus was identified as C. coccodes. To our knowledge, this is the first report of C. coccodes as the causal agent of black dot of potato in central Italy. We did not observe foliar outbreaks of the disease, which were reported from the United States (2). In both 1921 (1) and 1951 (3), the fungus was reported to cause severe outbreaks of the disease in northern Italy. Since then, its presence in Italy has been rarely recorded in potato (4). The occurrence of extremely dry and hot weather conditions during the summers of 1997 to 2000, which are favorable for disease development, made the disease particularly severe. We cannot exclude the possibility that the disease may have been present in central Italy before our observations, as it can be misdiagnosed and its symptoms can be masked by the symptoms of other diseases. The significance of black dot in central Italy needs to be reappraised in terms of both yield loss and tuber quality. References: (1) C. Arnaudi. Atti Ist. Bot. Univ. Pavia. Ser. 3, 1:71, 1924. (2) A. W. Barkdoll and J. R. Davis. Plant Dis. 76:131, 1992. (3) G. Goidanich. Inf. Fitopatol. 1:5, 1951. (4) S. Vitale et al. J. Plant Pathol. 80:265, 1998.

Occurrence of Pink Patch of Perennial Ryegrass Caused by Limonomyces roseipellis in Italy.

In March 1999, an unusual pink gelatinous mycelium was observed on several cultivars of perennial ryegrass (Lolium perenne L.) turf grown in experimental plots in Saint Andrea d'Agliano, Perugia (central Italy). Approximately 50% of the turf area showed symptoms on susceptible varieties. The same symptoms, although with lower severity, were observed during the following year in two experimental fields in northern Italy. The presence of mycelium on infected leaf blades was extensive during periods of high relative humidity and high temperature. The disease decreased progressively when weather conditions became dry and cold. A fungus, characterized by pink colonies, was consistently isolated from leaves of affected plants on potato dextrose agar (pH 5.5). On the basis of the presence of clamp connections and binucleate hyphal cells, the fungus was identified as Limonomyces roseipellis Stalpers & Loerakker, the causal agent of pink patch of turfgrass (1,3). For the pathogenicity test, one isolate of L. roseipellis was grown on maize flour and sand medium (2) at 22 ± 2°C for 14 days. Inoculum (20 g) was added to a sterile mixture of sand and peat moss (1:1; 640 g). Two hundred seeds of L. perenne (cv. Amadeus) were sown in boxes containing infested or noninfested soil as a control. Boxes were kept in a greenhouse at 22 ± 2°C, 80% relative humidity, and 14 h of sunlight per day. Four to five weeks after sowing, typical lesions resembling natural symptoms were observed only on plants grown in inoculum-infested soil, and L. roseipellis was consistently reisolated from diseased plants. Pink patch is probably underestimated in turf since the symptoms are less severe compared with red thread caused by Laetisaria fuciformis (McAlpine) Burdsall, and the development of mycelium of Limonomyces roseipellis is slower. References: (1) J. D. Kaplan and N. Jackson. Plant Dis. 67:159, 1983. (2) Y. L. Nene et al. ICRISAT Inf. Bull. 10:1, 1981. (3) J. A. Stalpers and W. M. Loerakker. Can. J. Bot. 60:529, 1982.

Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato.

A reproducible and accurate procedure, based on HPLC analysis, has been developed to determine simultaneously acibenzolar-S-methyl (CGA 245 704) and its acid derivative (CGA 210 007) in tomato leaves. The limit of detection and quantification of the method are 0.015 and 0.15 mg litre-1 for CGA 245 704 and 0.030 and 0.30 mg litre-1 for CGA 210 007. In tomato plants treated with 250 microM CGA 245 704, it was found that the inducer rapidly translocates from treated leaves (cotyledons, 1st and 2nd) to untreated leaves (3rd to 5th), with the maximum translocation (40% of the total quantity found) occurring 8 h after the treatment. CGA 245 704 residues decreased as time elapsed in both treated and untreated tomato leaves, reaching negligible values 72 h after treatment. The acid derivative, CGA 210 007, was formed in tomato plants as early as 2 h after CGA 245 704 treatment, albeit only in the treated leaves. CGA 210 007 residues decreased in treated tomato leaves with a trend similar to that observed for CGA 245 704. Treatment of tomato plants with CGA 245 704 or CGA 210 007 at 250 microM systemically protected the plants against Pseudomonas syringae pv tomato attacks, the causal agent of bacterial speak disease. Evidence of this were reductions in the degree of infection, the bacterial lesion diameter and the bacterial growth in planta. Since neither CGA 245 704 nor CGA 210 007 inhibited bacterial growth in vitro and the protection against bacterial speak of tomato was observed when the two compounds were completely degraded, the protection must be due to the activation of the plant's defence mechanisms.

First Report of Fusarium oxysporum f. sp. lycopersici Race 2 on Tomato in Italy.

During the summer of 1997, symptoms of Fusarium wilt were observed on tomato (Lycopersicon esculentum Mill.) cvs. Monica F1 and PS 110, which bear the I gene for resistance to race 1 of Fusarium oxysporum Schlechtend.:Fr. f. sp. lycopersici (Sacc.) W.C. Snyder & H.N. Hans., in two commercial production greenhouses in Latium (Fondi) and one greenhouse in Sardinia (Oristano). Infected plants showed yellowing, stunting, vascular discoloration, and premature death. A fungus from tomato stems with discolored vascular tissue was consistently isolated on potato dextrose agar (PDA) and, based on morphological features, was identified as F. oxysporum. To verify the pathogenicity of four fungal isolates, cv. Bonny Best tomato plants, which do not carry genes for Fusarium wilt resistance, were inoculated by dipping roots of 2-week-old seedlings in a suspension of 105 microconidia per ml for 30 s. Inocula were obtained from 1-week-old fungal cultures grown on PDA. Roots of control plants were dipped in water. Seedlings were transplanted to pots containing peat and river sand (1:1, vol/vol) and placed in a greenhouse at 20 to 25°C. One month after inoculation, all fungal isolates provoked wilting of inoculated plants. No symptoms were observed on control plants. The morphological features of the fungus reisolated from diseased plants were similar to those of the original isolates. Based on the pathogenicity test, we concluded that the fungal isolates belong to F. oxysporum f. sp. lycopersici. To determine the races of the fungal isolates, differential tomato lines VFN8 (I gene for resistance to race 1), Florida MH-1 (I and I2 genes for resistance to races 1 and 2), and I3R (I, I2, and I3 genes for resistance to races 1, 2, and 3) were inoculated with the four fungal isolates, using the same procedure described for the pathogenicity test. Because disease symptoms were detected on VFN8 but not on Florida MH-1 and I3R, we deduced that the fungal isolates belong to F. oxysporum race 2. This is the first report of F. oxysporum f. sp. lycopersici race 2 in Italy. Previous research indicated that race 1 is present in Italy (1). Currently, many commercially acceptable cultivars resistant to races 1 and 2 are available to Italian greenhouse growers. Reference: (1) M. Cirulli. Phytopathol. Mediterr. 4:63, 1965.

First Report of Lentil Ascochyta Blight Caused by Ascochyta lentis in Italy.

In May 1997, ascochyta blight incited by Ascochyta lentis Vassiljevsky was observed at an incidence of less than 5% in lentil (Lens culinaris Medik.) fields in Umbria (Central Italy). Symptoms appeared on leaves and stems as tan spots surrounded by a dark margin. Small black pycnidia that produced a pink exudate containing hyaline, 1 septate, 14.2 to 15.8 × 3.5 µm conidia under high humidity were visible in the center of the spots. The fungus was consistently isolated on potato dextrose agar from diseased leaves or stems. To satisfy Koch's postulates, a conidial suspension (106 conidia per ml) of the fungus was sprayed on leaves of 20-day-old lentil plants (landrace Castelluccio) that were maintained in a humidity chamber for 96 h after inoculation. Lesions resembling symptoms that occurred in the field were observed on plants 3 weeks after inoculation. Symptoms were not observed on control plants sprayed with water. The fungus reisolated from the diseased plants was identical to the original isolates. Based on morphological characteristics of pycnidia and conidia as well as pathogenicity, the fungus was identified as A. lentis. A deep-freeze blotter method (2) was used to detect A. lentis in lentil seeds of 20 local landraces used by Umbrian farmers and two accessions from Canada and Turkey, as well as in seed collected from infected fields. The fungus was present only in the two lentil accessions with an incidence of about 5%. Although the fungus had been isolated from Italian seed germplasm in 1986 (1), this is the first report of ascochyta blight occurring in lentil crops in Italy. The heavy rainfalls that characterize the first stage of lentil cultivation in Umbria are favorable for disease development while hot and dry conditions that usually occur during flowering and maturation prevent the dissemination of inoculum and the infection of the seeds. For these reasons, some Umbrian areas could be more suitable for production of ascochyta-free lentil seeds. References: (1) W. J. Kaiser and R. M. Hannan. Phytopathology 76:355, 1986. (2) T. Limonard. Proc. Int. Seed Test. Assoc. 33:343, 1968.