Paraphoma garibaldii sp. nov. causing leaf spot disease of Campanula rapunculoides in Italy.

Leaf and stem spots are among the most important diseases compromising ornamental plants worldwide. In this study, Paraphoma garibaldii sp. nov. is described from leaf lesions on Campanula rapunculoides in Piedmont, Northern Italy. The new species was characterised using a polyphasic approach including morphological characterisation and a multilocus molecular phylogenetic analysis based on partial nucleotide sequences of the translation elongation factor 1-α (tef1), the internal transcribed spacers (ITS) region and the ß-tubulin (tub2) markers. Pathogenicity tests and the fulfilment of Koch's postulates confirm P. garibaldii as a novel foliar pathogen of Campanula rapunculoides. Presently, the fungal infection due to Paraphoma garibaldii is known from a single location in Italy, and further surveys are required to determine its distribution and relative importance. Citation: Guarnaccia V, Martino I, Tabone G, Crous PW, Gullino ML (2022). Paraphoma garibaldii sp. nov. causing leaf spot disease of Campanula rapunculoides in Italy. Fungal Systematics and Evolution 9: 19-26. doi: 10.3114/fuse.2022.09.03.

Improving sustainable hydrogen production from green waste: [FeFe]-hydrogenases quantitative gene expression RT-qPCR analysis in presence of autochthonous consortia.

BACKGROUND: Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial consortium as an already selected source of implementation for biomass degradation and hydrogen production. The biocatalysts investigated as mainly relevant for hydrogen production were the [FeFe]-hydrogenases expressed in Clostridia, given their very high turnover rates. RESULTS: Bio-hydrogen accumulation was related to the modulation of gene expression of multiple [FeFe]-hydrogenases from two strains (Clostridium beijerinckii AM2 and Clostridium tyrobutyricum AM6) isolated from the same waste. Reverse Transcriptase quantitative PCR (RT-qPCR) was applied over a period of 288 h and the RT-qPCR results showed that C. beijerinckii AM2 prevailed over C. tyrobutyricum AM6 and a high expression modulation of the 6 different [FeFe]-hydrogenase genes of C. beijerinckii in the first 23 h was observed, sustaining cumulative hydrogen production of 0.6 to 1.2 ml H2/g VS (volatile solids). These results are promising in terms of hydrogen yields, given that no pre-treatment was applied, and suggested a complex cellular regulation, linking the performance of dark fermentation with key functional genes involved in bio-H2 production in presence of the autochthonous consortium, with different roles, time, and mode of expression of the involved hydrogenases. CONCLUSIONS: An applicative outcome of the hydrogenases genes quantitative expression analysis can be foreseen in optimising (on the basis of the acquired functional data) hydrogen production from a nutrient-poor green waste and/or low added value compost, in a perspective of circular bioeconomy.

Influence of different biological control agents and compost on total and nitrification-driven microbial communities at rhizosphere and soil level in a lettuce - Fusarium oxysporum f. sp. lactucae pathosystem.

AIMS: The response of rhizosphere and bulk soil indigenous microbial communities focusing on nitrifiers was evaluated after the application of different biological control agents (BCAs; Bacillus, Trichoderma, Pseudomonas) and compost in controlling lettuce Fusarium wilt. METHODS AND RESULTS: Experiments were conducted 'in situ' over two lettuce cropping seasons. Total fungal, bacterial and archaeal populations and the nitrifiers were analysed using quantitative polymerase chain reaction method. The pathogen, Fusarium oxysporum forma specialis lactucae (FOL), Bacillus, Trichoderma and Pseudomonas and three antifungal genes (chiA, 2,4-diacetylphloroglucinol - phlD and HCN synthase - hcnAB genes) were also assessed. Quantitative data were corroborated with disease severity (DS), potential nitrification activity and soil chemical parameters. The application of BCAs and compost resulted in the disease reduction by as much as 69%, confirmed by significant negative correlations between Bacillus subtilis, Trichoderma and Pseudomonas sp. abundances and DS. The FOL presence in the untreated control resulted in the nitrifiers niche differentiation. CONCLUSIONS: The used treatments were efficient against Fusarium wilt and did not influence negatively the nontarget microbial communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of BCAs and compost appears as an effective and safe strategy to implement sustainable agricultural practices.

Evolution of fungal populations in corn silage conserved under polyethylene or biodegradable films.

AIMS: To identify cultivable filamentous fungi before ensiling, after silage conservation, in farm-silos covered with two different plastic films (polyethylene (PE) vs biodegradable (MB)), as well as after aerobic exposure of whole-crop corn silage. METHODS AND RESULTS: Molecular techniques coupled with traditional microbial counting were utilized to identify the predominant fungal species. The cultivable fungal population changed remarkably from harvesting to silo opening. Anaerobiosis and low pH reduced mould count and the presence of Fusarium species both under PE and MB film. However, in the peripheral areas of the silo, where air penetration could not be completely prevented, the fungal population did not decrease. The predominant fungal species after aerobic exposure of silage was Aspergillus fumigatus, without differences between the two plastic films. CONCLUSIONS: Maintenance of anaerobiosis and a low pH also in the upper layer of the silo reduce the risk of mould growth during corn silage feed-out. SIGNIFICANCE AND IMPACT OF THE STUDY: Even if the new MB plastic film did not completely maintain the anaerobiosis in the upper layer of silage, the overall silage quality was not compromised and was similar to that observed under PE, indicating that the development and use of MB film to cover silage is promising, but needs some improvement.

PRE-PLANTING TREATMENTS WITH PHOSPHITE-BASED PRODUCTS AGAINST DIFFERENT FOLIAR AND SOIL-BORNE PATHOGENS OF VEGETABLE CROPS.

Fifteen experimental trials were carried out under greenhouse conditions to evaluate the efficacy of preventative treatments based on phosphite salts on the following pathosystems: tomato/Phytophthora nicotianae, zucchini/P. capsici, lettuce/Fusarium oxysporum f.sp. Iactucae, rocket/Fusarium oxysporum f. sp. raphani, wild rocket/Plectosphaerella cucumerina and basii/Peronospora belbahrii. The possible use of phosphite salts in nursery cultivation systems is considered in comparison with chemical fungicides. Phosphites-based products reduced 66-88% and 56-72% the severity of Phytophthora crown root rot of tomato and zucchini, respectively. Four application with the phosphites-based products provided a disease reduction of Fusarium wilt of lettuce from of 33 to 83% and of 45 to 68% on cultivated rocket. These products provide the most constant results when applied in three treatments against Plectosphaerella cucumerina with a disease reduction ranging between 34%-82%. Phosphite-based products showed results statistically similar to mefenoxam when tested against downy mildew of basil. Their contribution to disease management can be very interesting, because they can complement other control measures.

EFFECT OF BIOSOLARISATION ON THE MICROBIAL POPULATIONS OF SUBSTRATES INFESTED WITH FUSARIUM OXYSPORUM BY PCR-DGGE.

Biosolarisation consists of combining solarisation and organic matter application for controlling soilborne pathogens. The effects of this control strategy on the microbial community is almost unknown and needs to be investigated with molecular tools. The aim of the research was to investigate how biosolarisation can affect the structure of the microbial populations evaluated by a culture independent method using DGGE of PCR-amplified 18S-ITS genes-coding fragments from DNA extracted directly from infested substrate. Substrate samples were artificially infested with Fusarium oxysporum f. sp. conglutinans (FOC) and F. oxysporum f.sp. basilici (FOB) in order to evaluate the shift in fungal population by using culture independent methods. Solarisation was carried out with transparent polyethylene film during the summer period in a greenhouse located in Northern Italy, in combination or not with Brassica carinata defatted seed meals and/or compost. Biosolarisation treatment was carried out in a growth chamber by heating the substrate for 7 and 14 days at optimal (55-52 degrees C for 6 h, 50-48 degrees C for 8 h and 47-45 degrees C for 10 h/day) and sub-optimal (50-48 degrees C for 20 h, 45-43 degrees C for 8 h and 40-38 degrees C for 10 h/day) temperatures. Plate counts and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analyses were performed to evaluate the effect of biosolarisation on the microbial population. The abundance of FOC and FOB was reduced as a consequence of biosolarisation, while bacterial populations were higher compared to control samples during the experiment. PCR-DGGE fingerprints of the ascomycete community obtained from DNA directly extracted from infested substrate samples showed that the use of organic amendments increased the similarity of the fungal populations.

First Report of Web Blight on Rose Campion (Lychnis coronaria) Caused by Rhizoctonia solani AG 1-IB in Italy.

Lychnis coronaria (syn. Silene coronaria), rose campion, is a perennial in the Caryophyllaceae used in gardens. In the summer of 2014, a web blight was observed in a private garden located near Biella (northern Italy), approximately 45°39'N 8°00'E, on 40% of 100 5-month-old plants grown in sandy soil. In the days preceding the outbreak of the disease, daytime temperatures ranged from 18 to 24°C and relative humidity from 45 to 83%. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Under conditions of high relative humidity, a scant amount of whitish mycelium developed on leaves of about 50% of diseased plants. Eventually, infected plants died about 10 days after symptoms appeared. Symptomatic tissues of stems and leaves were disinfected for 10 s in 1% NaOCl, rinsed in sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates were paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 4, AG 7, and AG 11) (2) and examined microscopically. The Lychnis isolates anastomosed only with the AG 1 tester strain, with low fusion frequency. The anastomosis point was obvious: the hyphal diameter at the point of anastomosis was reduced and death of adjacent cells was observed, indicating an anastomosis reaction (1). Mycelium maintained on PDA at 23 ± 1°C was coarse and reddish brown. After 5 days of growth, mycelium started differentiating numerous sclerotia, often aggregated. Mature sclerotia were dark, spheroidal, with diameters ranging from 0.2 to 1.6 (average 0.6) mm. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 609-bp amplicon (GenBank Accession No. KM596899) showed a 98% homology with the sequence of the R. solani isolate FJ746937 obtained from Zoysiagrass. On the basis of molecular and cultural characteristics and anastomosis tests, the isolates from L. coronaria were identified as R. solani AG 1-IB (4). For pathogenicity tests performed in August, mycelial disks (8 mm diam.) from 10-day-old PDA cultures of an isolate of the fungus were placed on four healthy 6-month-old L. coronaria plants (four stem and six leaf disks per plant). Four plants inoculated with disks of PDA served as controls. Plants were covered with plastic bags for 4 days and maintained in a garden located in the same area in which the disease appeared, at field temperatures ranging from 15 to 28°C. The first symptoms developed 4 days after inoculation, and 15 days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated from the stem of symptomatic plants, whereas no colonies developed from controls, which all remained healthy. This is the first report of blight of L. coronaria caused by R. solani in Italy or anywhere else in the world. The impact of this disease may become a significant problem for L. coronaria, a very common species in Italian gardens. References: (1) D. E. Carling. Page 37 in: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers, The Netherlands, 1996. (2) A. Ogoshi. Ann. Rev. Phytopathol. 25:125, 1987. (3) B. Sneh et al. Identification of Rhizoctonia species. APS Press, St. Paul, MN, 1991. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.

Phytophthora cryptoea on Common Sage (Salvia officinalis L.) in Italy.

During the spring of 2013, many plants of common sage (Salvia officinalis L.), grown as potted plants in a commercial farm at Albenga (northern Italy) showed extensive symptoms of foliar wilt and root rot. The first symptoms developed with temperatures ranging between 8 and 26.5°C, average 17°C, and consisted of leaf chlorosis, wilting, and collapse. Severe root and crown rot were also observed, leading to sudden collapse of approximately 60% of the 6,000 plants within 60 days from transplant. Symptomatic tissues from the root and collar of infected plants were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in water, and placed on a selective medium for oomycetes (3). A Phytophthora-like organism (1) was consistently isolated and was transferred to carrot agar. Mycelial disks of the isolate DB13GIU02 were floated in petri plates containing soil extract (1), under continuous fluorescent light at room temperature. Hyphal swelling was abundant in such aqueous medium, measuring 6.4 to 20.1 (13.1 average) µm. Sporangia were obpyriform, persistent, and nonpapillate, measuring 25.3 to 55.1 × 17.9 to 37.1 (average 42.8 to 27.9) µm. Oospores and chlamydospores were absent. The same isolate was tested with two isolates of P. cryptogea from Quercus ilex (PH050, mating type A1) and from Pistacia lentiscus (PH017, mating type A2) on carrot agar, at 23 ± 1°C in the dark. Only the paring of DB13GIU02 with PH017 was successful and produced oogonia with diameter of 28.3 to 34.6 (average 31.7) µm, oospores with diameter of 28.0 to 32.2 (average 29.2) µm, and anphigynous antheridia of 10.5 to 15.1 × 11.6 to 15.1 (average 13.5 × 13.3) µm. DNA of the three isolates was extracted by using the Nucleospin Plant kit (Macherey Nagel). PCR of DNA amplified with primers Cryp 1 and Cryp 2 (4) from all P. cryptogea isolates produced a specific amplicon. The internal transcribed spacer (ITS) region of rDNA of the isolate DB13GIU02 was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis of the 845-bp segment (GenBank Accession No. KM458193) showed a 99% homology with the sequence of P. cryptogea GU111631. Pathogenicity tests were performed on healthy common sage 60-day-old plants by using one strain of P. cryptogea grown on a mixture of 2:1 wheat/hemp kernels. Infested kernels (10 g/liter of substrate) were mixed into a steam-disinfested substrate based on sphagnum peat/pomix/pine bark/clay (50:20:20:10 v/v). Control plants were treated with uninoculated wheat/hemp kernels mixed into the steam-disinfested soil. The trial was repeated once. Fifteen plants per treatment were used. All plants were kept in a growth chamber at 20 ± 1°C. Inoculated plants became chlorotic 7 days after inoculation, and root and crown rot developed 15 days after inoculation. P. cryptogea was consistently reisolated from inoculated plants. No colonies were isolated on the selective medium from control plants that remained symptomless. P. cryptogea has been reported on S. officinalis in the United States (2), while in Italy the same pathogen has been observed on S. leucantha. This is the first report of P. cryptogea on S. officinalis in Italy. The economic importance of the disease can increase due to the expanding use of this plant both as an aromatic for culinary purposes and for landscaping. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St. Paul, MN, 1996. (2) S. T. Koike et al. Plant Dis. 81:959, 1997. (3) H. Masago et al. Phytopathology 67:25, 1977. (4) D. Minerdi et al. Eur. J. Plant Pathol. 122:227, 2008.

Root Rot of Spinach in Southern Italy Caused by Pythium aphanidermatum.

During the spring of 2014, spinach (Spinacia oleracea L.) plants of the cv. Crocodile (Rijk Zwaan, De Lier, The Netherlands), grown in a clay loam soil under commercial greenhouse conditions near Salerno (southern Italy), showed stunting, extensive chlorosis, and root rot. Plants were irrigated by overhead sprinklers using well water. Symptoms first developed 20 days after sowing, at air temperatures of 23 to 30°C, and 35% of plants (approximately 15 million plants in 10 ha) were affected. Roots were severely affected, appeared water-soaked and brown, and were characterized by a soft rot. Eventually, affected plants wilted and collapsed. Fifty fragments, each 1 mm2, were excised from symptomatic roots of 10 plants, dipped in a solution containing 1% sodium hypochlorite, rinsed in sterilized water, dried on sterilized paper towel, and plated on both potato dextrose agar (PDA) and the medium BNPRA, which is semi-selective for oomycetes (3). After 5 days of incubation under constant fluorescent light at 22 ± 1°C, 80% of the root sections developed oomycete colonies. One representative isolate, grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup; 15 g agar; 0.5 g CaCO3; 1 liter distilled water) and observed with a light microscope, showed aseptate hyphae 3.3 to 6.5 (mean 5.5) µm wide. Oogonia were globose, smooth, and 22.2 to 31.0 (average 26.3) µm in diameter. Antheridia were barrel-shaped, while oospores were globose and 17.3 to 22.6 (mean 20.9) µm in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of this isolate was amplified using ITS1/ITS4 primers and sequenced. BLAST analysis (1) of the 647-bp segment showed 100% homology with ITS sequences of Pythium aphanidermatum in GenBank (Accession Nos. KJ755088.1, KJ162355.1, KF840479, and KF561235.1). The nucleotide sequence for the Italian spinach isolate was assigned No. KM111256. Pathogenicity tests were performed twice on 20-day-old spinach plants of the cv. Merlo (L'Ortolano, Cesena, Italy), grown in 2-liter pots in a steam-disinfested organic peat substrate (black peat, pH 6.5 to 6.8, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) moistened to field capacity, and infested with wheat and hemp kernels colonized with isolate Py 1-14 of P. aphanidermatum at 1 g/liter. Five plants were transplanted into each of four pots filled with infested peat, while the same number of plants was grown in non-infested substrate as a control. Plants were kept in two growth chambers, each with 12 h of light per day at 20 or 30°C, and were irrigated daily to maintain the potting medium at field capacity. Symptoms first developed 5 days after the spinach was transplanted into infested potting medium in the growth chamber maintained at 30°C. After 10 days, all plants in this growth chamber were dead, while only 5% of the plants growing in infested potting medium in the 20°C growth chamber were affected. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in infested medium. No fungi were re-isolated from the asymptomatic control plants grown in non-infested substrate. To our knowledge, this is the first report of P. aphanidermatum causing root rot on S. oleracea in Italy. The same pathogen has been reported to cause root rot of spinach in other countries, including the United States (2). The disease is, at present, limited to the affected greenhouses observed in this study. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. L. Bates and M. E. Stanghellini. Plant Dis. 68:989, 1984. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, FL., 2002.

VARIETAL RESISTANCE TO CONTROL FUSARIUM WILTS OF LEAFY VEGETABLES UNDER GREENHOUSE.

Fusarium wilts of leafy vegetables are difficult to manage under intensive cropping systems. The objective of this study was to evaluate, in three experimental trials, the susceptibility of commercial cultivars of lettuce, wild and cultivated rocket and lamb's lettuce to Fusarium wilts in orderto provide information to breeders, as well as to growers. Some of the cultivars of lettuce tested were completely resistant to the three races of Fusarium wilt. It is interesting to observe that most of the resistant cultivars were 'Batavia red'. Only few rocket cultivars commercially available show a partial resistant reaction to F. oxysporum f.sp. raphani, while, varietal resistance is not applicable at the moment to control Fusarium wilt of lamb's lettuce. The integration of cultural practices, use of resistant cultivars, when available, chemicals and biological control agents, permit to prevent and manage these important diseases on leafy vegetables for fresh-cut production.

CONTROL OF SOIL-BORNE DISEASES BY DIFFERENT COMPOSTS IN POTTED VEGETABLE CROPS.

The composting process and the type and nature of wastes and raw materials influence the maturity, quality and suppressiveness of composts. Variability in disease suppression also depends on the pathosystem, on soil or substrate type, on chemical-physical conditions, like pH and moisture, and on the microbial component of compost. The aim of the research was to evaluate the suppressiveness of composts, originated from green wastes and/or municipal biowastes, and produced by different composting plants located in Europe. The composts were tested against soil-borne pathogens in greenhouse on potted plants: Fusarium oxysporum f.sp. busilici/basil, Pythium ultimum/cucumber, Rhizoctonia solani/bean. Composts were blended with a peat substrate at different dosages (10, 20 and 50% vol./vol.) 14 days before seeding or transplanting. Pythium ultimum and Rhizoctonia solani were mixed into the substrate at 0.5 g of wheat kernels L(-1) 7 days before seeding, while, in the case of Fusarium oxysporum f.sp. basilici, chlamydospores were applied at 1 x 10(4) CFU/g. Seeds of basil, cucumber and bean were sown into 2 L pots in greenhouse. The number of alive plants was counted and above ground biomass was weighed 30 days after seeding. The number of infected cucumber and basil plants was significantly reduced by increasing dosages of composts, but municipal compost was phytotoxic when applied at high dosages compared to green compost. Moreover, municipal compost increased the disease caused by Rhizoctonia solani on bean. The use of compost in substrates can be a suitable strategy for controlling soil-borne diseases on vegetable crops, but results depend on type of composts, application rates and pathosystems.

First Report of Dry and Soft Rot of Cereus marginatus var. cristata Caused by Fusarium oxysporum in Italy.

During the winter of 2013, 50% of 20,000 plants of Cereus marginatus var. cristata, Cactaceae family, grown in a commercial farm located in Liguria (northern Italy) showed symptoms of a dry or soft rot. In the case of dry rot, affected plants showed on the stem superficial necrosis and dry rot, irregularly shaped, 1 to 10 mm, while epidermal and cortical tissues were wounded. Affected plants survived but they lost ornamental value. In the case of soft rot, associated with conditions of higher relative humidity, rots on the stem extended as far as 4 cm in width. The internal part of bark, cambium, and xylem tissues as far as about 3 cm in depth was rotted. Vascular tissues were not discolored. Plants died in about 20 days. A Fusarium sp. was consistently isolated from symptomatic tissue on Komada selective medium (2) from plants showing soft rot. The isolates were purified and subcultured on potato dextrose agar (PDA). On PDA, the cultures produced a thick and soft growth of white to light pink mycelium and pale pink pigments in the agar. On Spezieller Nährstoffarmer agar (SNA), cultures produced short monophialides with unicellular, ovoid-elliptical microconidia measuring 3.7 to 8.2 × 1.7 to 3.5 (average 5.4 × 2.5) µm. On carnation leaf-piece agar (CLA), chlamydospores were abundant, terminal or intercalary, single or paired, but frequently also aggregated. On the same medium, at temperatures ranging from 20 to 24°C (14 h daylight, 10 h dark), cultures produced light orange sporodochia with macroconidia. These were 3 to 4 (sometimes 5) septate, nearly straight with a foot-shaped basal cell and a short apical cell, and measured 28.5 to 41.4 × 3.3 to 4.9 (average 35.0 × 4.0) µm. Such characteristics are typical of Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen (3). Amplification of the internal transcribed spacer (ITS) of the rDNA using primers ITS1/ITS4 yielded a 504-bp amplicon (GenBank Accession No. KJ909935). Sequencing and BLASTn analysis of this amplicon showed a 100% homology with the sequence of F. oxysporum KC304802. To confirm pathogenicity, two Fusarium isolates were tested. For each isolate, three 2-year-old healthy plants of C. marginatus were inoculated by introducing into lesions (4 lesions/plant) artificially produced on the stem sterile needles contaminated with the pathogen (4). Inoculum was obtained from pure cultures grown on PDA. Control plants were punctured with sterile needles without inoculum. All the plants were placed in a greenhouse, at temperatures ranging between 16 and 24°C. For both tested strains, the first necrosis of stem tissues developed around the needles 7 days after the artificial inoculation, while non-inoculated plants remained healthy. Then, necrosis extended causing soft rot on plants maintained at relative humidity ranging from 55 to 65%. F. oxysporum identified by morphological characteristics was consistently isolated from symptomatic plants. The pathogenicity test was conducted twice. F. oxysporum has been reported on Cereus sp. in the United States and on C. peruvianus monstruosus in Italy (1). Currently, this disease is present in a few commercial nurseries in Liguria, although it could spread further and cause important economic losses. References: (1) A. Garibaldi et al. Plant Dis. 95:877, 2011. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (4) V. Talgø and A. Stensvand. OEPP/EPPO Bulletin 43:276, 2013.

First Report of Web Blight on Rebutia perplexa Caused by Rhizoctonia solani AG 2-2-IIIB in Italy.

Rebutia perplexa, Cactaceae family, is a clumping fine thorny cactus, producing several flushes of pink flowers. In the spring of 2013, a blight was observed in a farm located near Imperia (northern Italy) on 2% of 2,000 3-year-old plants, grown in plastic pots. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Flowers also rotted and wilted. In the presence of high relative humidity, a rare, whitish mycelium developed on the surface of the substrate. Eventually, infected plants died. Symptomatic tissues of the stem were taken from 10 plants and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates obtained from affected plants were successfully paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 2-2-IV, AG 4, AG 7, AG 11) (2) and examined microscopically. Three replicated pairings were made for each tester strain. The Rebutia isolates anastomosed only with AG 2-2-IIIB tester strain with high hyphal fusion frequency. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and death of adjacent cells was observed, indicating anastomosis reactions (1). Tests were performed twice. Mycelium of 15-day-old isolates maintained at 27 to 30°C, appeared whitish or pale buff in color, coarse, with a concentric zonation, scarce aerial mycelium, and without sclerotia. The optimum temperature for mycelium growth was 30°C (daily growth rate: 24.6 mm) and isolates grew also at 35°C. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 523-bp amplicon (GenBank Accession No. KF719318) showed a 99% homology with the sequence of the R. solani AG 2-2-IIIB isolate GU811672. The nucleotide sequence has been assigned the GenBank Accession No. KF719318. Therefore, on the basis of molecular characteristics, anastomosis tests, temperature growth, and cultural characteristics, the isolates from R. perplexa were identified as R. solani AG 2-2-IIIB. For pathogenicity tests, 3 g of colonized wheat kernel from 10-day-old cultures of a representative isolate of the fungus was added per 1 l of substrate in 12 potted healthy plants of R. perplexa. The inoculum was prepared by inoculating wheat kernels with the mycelium of 10-day-old cultures of the fungus and incubating at 25 ± 1°C (12 h fluorescent light, 12 h dark). Twelve plants inoculated with non-infested wheat kernels served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 25 ± 1°C. The first symptoms, similar to those observed in the farm, developed 5 days after inoculation. Fifteen days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated only from the stems of symptomatic plants. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. This is, to our knowledge, the first report of blight of R. perplexa caused by R. solani in Italy as well as worldwide. References: (1) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease control. Kluwer Academic Publishers, The Netherlands, pp. 37-47, 1996. (2) A. Ogoshi. Ann. Rev. Phytopathol. 25:125, 1987. (3) B. Sneh et al. Identification of Rhizoctonia species. APS Press, St Paul, MN, 1991.

First Report of Stem Rot on Cereus peruvianus monstruosus Caused by Bipolaris cactivora (Petr.) Alcorn in Italy.

Cereus peruvianus monstruosus, known as "monster cactus," family Cactaceae, is grown as a potted plant. In the winter of 2013, a stem rot was observed on a farm located near Ventimiglia (northern Italy) on 80% of 4,000 9-month-old plants grown in trays in a peat substrate. Symptoms consisted of a rapid rot of the upper portion of the stem. Affected stems at first showed yellowish spots that became brown irregular necrotic lesions with well-defined margins. The tissues below the affected areas were blackened and dry but became soft in the presence of high relative humidity. Fungal sporulation on rotted tissues consisted of caespitose, non-branched, septate conidiophores, olivaceous to brown at the base, paler above, measuring 89.0 to 196.9 × 6.2 to 8.7 (average 124.8 × 7.0) µm. Single conidia were borne on terminal cells. At maturity, conidia with 2 to 5 (average 3) septa were brownish-olivaceous, varying in shape from obclavate, fusiform, ellipsoid or sometimes furcate, and measuring 23.4 to 48.6 × 8.0 to 12.6 (average 38.8 × 10.3) µm. Symptomatic tissues were immersed in 1% sodium hypochlorite for 2 to 3 s and rinsed in sterile distilled water, then fragments excised from the margin of internal lesions were cultured on potato dextrose agar (PDA) medium amended with 25 mg/l of streptomycin sulfate and incubated at 20 to 23°C under alternating daylight and darkness (10 h light, 14 h dark). A fungus that was consistently isolated was subcultured on PDA. At maturity, dark green floccose colonies comprised of light brown septate hyphae, 4.2 to 8.1 (average 5.6) µm in width, produced non-branched, pale to dark brown, septate conidiophores, measuring 99.6 to 176.1 × 4.5 to 6.5 (average 146.7 × 5.4) µm. The conidia produced on PDA were similar to those observed on infected tissues and measured 20.6 to 40.7 × 7.5 to 11.4 (average 32.0 × 9.7) µm, with 1 to 3 septa (average 2). On the basis of the morphological characteristics, the fungus was identified as Bipolaris cactivora (Petr.) Alcorn [Syn.: Drechslera cactivora (Petr.) M. B. Ellis] (4). The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was amplified for one isolate using ITS1/ITS4 primers and sequenced (GenBank Accession No. KF041822). BLASTn analysis (1) of the 557-bp segment showed a 99% similarity with the ITS sequence of Bipolaris cactivora HM598679. For pathogenicity tests, 8 mm diameter mycelial disks removed from 15-day-old PDA cultures of the fungus were placed at the wounded stem apexes of three 7-month-old healthy plants (three disks per plant). Three plants inoculated with non-inoculated PDA disks served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 23 ± 1°C with 12 h light/dark. By 8 days after inoculation, all the inoculated stems were rotted and 10 colonies of B. cactivora were re-isolated from infected tissues. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Several hosts are listed for B. cactivora including C. peruvianus, and the pathogen has been reported in the United States (2) and in South Korea (3). To our knowledge, this is the first report of B. cactivora on C. peruvianus monstruosus in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. APS Press, St Paul, MN, 1989. (3) I. H. Hyun et al. Res. Plant Dis. 7:56, 2001. (4) A. Sivanesan. Mycopathologia 111:125, 1990.

First Report of Root Rot Caused by Pythium aphanidermatum on Bell Pepper (Capsicum annuum L.) in Italy.

During July 2012, symptoms of root rot were observed on bell pepper (Capsicum annuum) grown in 2,000 m2 of commercial greenhouses near Cuneo in northern Italy. Symptoms first developed 30 to 40 days after transplanting, when greenhouse temperatures ranged from 25 to 30°C, and 10% of the plants were affected. Affected plants were stunted with leaf chlorosis, reduced growth, and sudden wilting. Roots were severely affected with a brown discoloration, water-soaking, and soft rot. Eventually, affected plants collapsed. Tissue fragments of 1 mm2 were excised from symptomatic roots, dipped in a 1% sodium hypochlorite solution, and placed on potato dextrose agar (PDA) and an agar medium selective for oomycetes (3). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. An isolate grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup, 15 g agar, 0.5 g CaCO3, and 1 liter distilled water) showed aseptate hyphae that were 3.5 to 6.3 µm (avg. 5.2 µm) wide. Oogonia were globose, smooth, and 24.3 to 29.0 (avg. 25.1) µm in diameter. Antheridia were barrel-shaped, while oospores were globose, and 17.3 to 23.5 µm (avg. 21.2 µm) in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The ITS region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 781-bp segment (GenBank Accession KF840479) showed 100% homology with the ITS sequence of an isolate of Pythium aphanidermatum in GenBank (AY598622.2). Pathogenicity tests were performed twice on 30-day-old plants of C. annuum cv. Cuneo grown in 2-L pots (4 plants/pot), containing a steam-disinfested, organic peat substrate (70% black peat and 30% white peat, pH 5.5 to 6.0, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) that was infested with wheat and hemp kernels colonized by the isolate of P. aphanidermatum, at a rate of 1 g colonized kernels/liter potting medium. The inoculum was prepared by autoclaving at 121°C for 30 min a mixture of wheat-hemp kernels (2:1 v/v) in a 1-liter flask, to which the bell pepper isolate of P. aphanidermatum was added in the form of colonized agar medium selective for oomycetes plugs. Before use, the inoculated flask was incubated for 10 days at 22°C in the dark. Four plants/pot were transplanted into each of four pots filled with the infested medium/growth chamber, while the same number of plants were grown in non-infested substrate in pots in each growth chamber. Plants were kept in two growth chambers, one set at 20°C and the other at 28°C. Symptoms first developed 7 days after inoculation. After 30 days, 50% of inoculated plants showed brown roots and died in the growth chamber set at 28°C, while only 10% of the plants were symptomatic at 20°C. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in the infested soil by using the same protocol as the original isolations, while no fungal colonies were obtained from asymptomatic roots of the non-inoculated control plants. To our knowledge, this is the first report of the presence of P. aphanidermatum on C. annuum in Italy. The same disease was reported in the United States (2). The importance of the disease, although limited in distribution at present to the greenhouses surveyed in northern Italy, could increase in areas where sweet pepper is grown intensively. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. O. Chellemi et al. Plant Dis. 84:1271, 2000. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Boca Raton, FL, 2002.