G. Gilardi

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.

First Report of Fusarium oxysporum on Lettuce in Europe

In spring 2001, plants of the lettuce cv. Salad Bowl showing symptoms of a wilt disease were observed in several commercial plastic greenhouses near Bergamo, in northern Italy. Wilted plants were first observed during the spring and summer of 2001 when temperatures were between 26 and 35°C. Symptoms were observed in the same area and the same farms in March 2002, in concomitance with a period of high temperatures. Although the distribution of the disease was generally uniform, symptoms were more severe in the central part of the greenhouses where temperatures were warmest. Symptoms were first observed at thinning, when seedlings (30 days old) appeared wilted. Vascular tissues of affected seedlings appeared red or brown. Affected plants were stunted and developed yellow leaves and brown or black streaks in the vascular system. The vascular streaks in the yellow leaves extended from the crown and were continuous with a red-brown discoloration in the vascular system of the crown and upper taproot. Symptoms were typically not visible on the outside of the crowns or roots. Fusarium oxysporum was consistently and readily isolated from symptomatic vascular tissues on a Fusarium-selective medium (2). Seeds of cv. Salad Bowl were planted in steam-sterilized soil artificially infested with 1 × 104 CFU/g soil of each of two isolates of F. oxysporum obtained from infected plants. Plants grown in noninfested soil served as noninoculated control treatments. Plants (25 per treatment) were grown at 25 to 28°C in growth chambers. Wilt symptoms and vascular discoloration of the roots, crown, and veins developed 20 days after seeding for all plants grown in the infested soil, and F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was conducted twice. To our knowledge, this is the first report of F. oxysporum on lettuce in Europe. A wilt of lettuce attributed to F. oxysporum f. sp. lactucae was reported in Japan in 1967 (3) and in the United States in 1993 (1), in the latter case the incitant organism was described as F. oxysporum f. sp. lactucum. References: (1) J. C. Hubbard and J. S. Gerik. Plant Dis. 77:750, 1993. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (3) T. Matuo and S. Motohashi. Trans. Mycol. Soc. Jpn. 8:13, 1967.

Seed transmission ofFusarium oxysporum f.sp.lactucae

Twenty-seven seed samples belonging to the lettuce cultivars most frequently grown in Lombardy (northwestern Italy), in an area severely affected by Fusarium wilt of lettuce, were assayed for the presence ofFusarium oxysporum on a Fusarium-selective medium. Isolations were carried out on subsamples of seeds (500 to 1500) belonging to the same seed lots used for sowing, and either unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates ofF. oxysporum obtained was tested in four trials carried out on lettuce cultivars of the butterhead type, very susceptible to Fusarium wilt. Nine of the 27 samples of seeds obtained from commercial seed lots used for sowing in fields affected by Fusarium wilt were contaminated byF. oxysporum. Among the 16 isolates ofF. oxysporum obtained, only one was isolated from disinfected seeds. Three of the isolates were pathogenic on the tested cultivars of lettuce, exhibiting a level of pathogenicity similar to that of the isolates ofF. oxysporum f.sp.lactucae obtained from infected wilted plants in Italy, USA and Taiwan, used as comparison. The results obtained indicate that lettuce seeds are a potential source of inoculum for Fusarium wilt of lettuce. The possibility of isolatingF. oxysporum f.sp.lactucae, although from a low percent of seeds, supports the hypothesis that the rapid spread of Fusarium wilt of lettuce observed recently in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed.

A review and critical analysis of the European situation of soilborne disease management in the vegetable sector

This review concentrates on the situation of soilborne disease management on several vegetable crops with the use of different strategies, including host resistance and grafting, with special focus—based on the experience gained in Italy and Europe—on the practical problems encountered after the phaseout of methyl bromide. Special attention is given to the emergence of new diseases or the re-emergence of old ones on several crops, and on the results and problems encountered by using grafting, biofumigation and other non-chemical control methods. The search for effective, economically and environmentally sound methods for soil and substrate disinfestation remains a continuous and challenging task for growers, despite intensive research. The European Regulation No 1107/2009, concerning the placing of plant protection products on the market and the European Directive No 2009/128/EC, establishing a framework for Community action to achieve the sustainable use of pesticides, requires, by 2014, that all professional users implement the general principles of IPM. Research efforts associated with phaseout of methyl bromide generated new knowledge about the biology, ecology and management of soilborne pathogens and suggested more attention towards crop and soil health.

Vegetative Compatibility Groups of Fusarium oxysporum f. sp. lactucae from Lettuce

Fusarium oxysporum f. sp. lactucae, the causal agent of Fusarium wilt of lettuce, has been reported in three continents in the last 10 years. Forty-seven isolates obtained from infected plants and seed in Italy, the United States, Japan, and Taiwan were evaluated for pathogenicity and vegetative compatibility. Chlorate-resistant, nitrate-nonutilizing mutants were used to determine genetic relatedness among isolates from different locations. Using the vegetative compatibility group (VCG) approach, all Italian and American isolates, type 2 Taiwanese isolates, and a Japanese race 1 were assigned to the major VCG 0300. Taiwanese isolates type 1 were assigned to VCG 0301. The hypothesis that propagules of Fusarium oxysporum f. sp. lactucae that caused epidemics on lettuce in 2001-02 in Italian fields might have spread via import and use of contaminated seeds is discussed.

Efficacy of the biocontrol agents Bacillus subtilis and Ampelomyces quisqualis applied in combination with fungicides against powdery mildew of zucchini

The activity of two biofungicides based on Bacillus subtilis and Ampelomyces quisqualis alone and in combination with fungicides against Podosphaera xanthii on zucchini was tested in five experimental trials carried out under greenhouse conditions. The population of P. xanthii used throughout the work for artificial inoculation was able to cause slight infections on zucchini plants treated with the field dosages of azoxystrobin and penconazole. B. subtilis when combined with azoxystrobin against P. xanthii at the recommended field dosages provided better powdery mildew control than the two control measures alone. Such results were consistent throughout the different trials. A similar synergistic effect was shown by the combination of A. quisqualis with myclobutanil: however, in this case, more studies are necessary to confirm the consistency of that phenomenon. The results obtained in this study support the possibility of a synergistic effect among the biocontrol agent B. subtilis and QoI fungicides. Such interaction is very interesting because of the presence of resistance towards QoI in the populations of P. xanthii.ZusammenfassungDie Wirkung der bakteriellen bzw. pilzlichen Antagonisten Bacillus subtilis und Ampelomyces quisqualis gegenüber einem der beiden bekannten Echten Mehltaupilze an Cucurbitaceen, Podosphaera xanthii, wurde an Zucchini einzeln oder kombiniert mit synthetischen Fungiziden in fünf unterschiedlichen Varianten im Gewächhaus untersucht. Die verwendete Population von P. xanthii konnte Zucchinipflanzen, die mit im Freiland üblichen Dosierungen von Azoxystrobin oder Penconazol behandelt waren, in relativ geringem Ausmaß infizieren. Die kombinierte Anwendung von B. subtilis und Azoxystrobin wirkte in allen fünf Varianten effizienter als beide Antagonisten einzeln. Eine vergleichbare, allerdings durch weitere Untersuchungen zu bestätigende Wirkung, zeigte die Kombination von A. quisqualis und Myclobutanil. Die Ergebnisse erhärten die Hypothese, dass B. subtilis und Strobilurine synergistisch gegenüber P. xanthii wirken, was im Hinblick auf Resistenzentwicklungen des Erregers gegenüber dieser Wirkstoffgruppe von größerem praktischen Interesse sein könnte.

Evidence for an expanded host range ofFusarium oxysporum f.sp.raphani

The pathogenicity of four isolates ofFusarium oxysporum obtained from infected cultivated rocket (Eruca vesicaria) and wild (sand) rocket (Diplotaxis tenuifolia) was tested on the following cruciferous hosts: stock, radish, wild and cultivated rockets, and various species in the cabbage tribe: cabbage (Brassica oleracea var.sabauda), cauliflower (Brassica oleracea var.botrytis), Brussels sprouts (Brassica oleracea var.gemmifera), broccoli (Brassica oleracea var.italica), turnip (Brassica rapa var.rapa). The results indicated that isolates ofF. oxysporum from cultivated and wild rocket belong to theforma specialis raphani. The isolates from rocket were pathogenic on cabbage, Brussels sprouts, broccoli, turnip, radish and stock; isolates ofF. oxysporum conglutinans from cabbage and radish, and the isolate ofF. oxysporum f.sp.raphani from rape obtained from the ATCC collection, were pathogenic on both cultivated and wild rocket.

Mycotoxin Production in Liquid Culture and on Plants Infected with Alternaria spp. Isolated from Rocket and Cabbage

Fungi belonging to the genus Alternaria are common pathogens of fruit and vegetables with some species able to produce secondary metabolites dangerous to human health. Twenty-eight Alternaria isolates from rocket and cabbage were investigated for their mycotoxin production. Five different Alternaria toxins were extracted from synthetic liquid media and from plant material (cabbage, cultivated rocket, cauliflower). A modified Czapek-Dox medium was used for the in vitro assay. Under these conditions, more than 80% of the isolates showed the ability to produce at least one mycotoxin, generally with higher levels for tenuazonic acid. However, the same isolates analyzed in vivo seemed to lose their ability to produce tenuazonic acid. For the other mycotoxins; alternariol, alternariol monomethyl ether, altenuene and tentoxin a good correlation between in vitro and in vivo production was observed. In vitro assay is a useful tool to predict the possible mycotoxin contamination under field and greenhouse conditions.

First report of Fusarium oxysporum on Eruca vesicaria and Diplotaxis spp. in Europe.

Two types of rocket are available on the market in Italy: (i) Eruca vesicaria (synonym E. sativa) known as ruchetta or cultivated garden rocket; and (ii) several species of Diplotaxis (Diplotaxis erucoides, D. muralis, and D. tenuifolia), which are wild plants now widely cultivated. Rocket is increasingly used in the mediterranean cuisine as salad and or to decorate dishes. In spring 2002, plants of the cultivated (E. vesicaria) and wild (Diplotaxis spp.) rocket showing symptoms of a wilt disease were observed in several commercial plastic greenhouses near Bergamo, in northern Italy. Wilted plants were first observed during the spring and summer of 2001 when temperatures were between 26 and 35°C. In May 2002, symptoms were again observed in the same area, on the same farm as well as other farms. Although the distribution of the disease was generally uniform, symptoms were more severe in the central part of the greenhouses where temperatures were warmest (30 to 60% of plants were affected). Diseased plants were stunted and chlorotic with brown or black streaks in the vascular system. Vascular tissues of affected plants appeared red or brown. Vascular streaks in the chlorotic leaves extended from the crown and were continuous with a red-brown discoloration in the vascular system of the crown and upper taproot. Fusarium oxysporum was consistently and readily isolated from symptomatic vascular tissues when plated on a Fusarium-selective medium (2). Microconidia measured 8.8 × 3.0 µm. E. vesicaria and Diplotaxis spp. were grown in steam-sterilized soil, and 10 days after emergence they were artificially inoculated by root dipping in a spore suspension (1 × 105 CFU/ml) of three F. oxysporum strains collected from infected plants. Uninoculated plants served as control. Plants (60 per treatment) were grown at 25 to 28°C in growth chambers. Wilt symptoms developed on all plants 20 days after inoculation, and F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was conducted twice. To our knowledge, this is the first report of F. oxysporum on cultivated rocket in Europe and the first on wild rocket (Diplotaxis spp.) in the world. A wilt of E. sativa attributed to F. oxysporum f. sp. erucae was previously reported in India in 1973 (1). Studies are being carried out to determine if the Italian isolates of F. oxysporum belong to the same formae speciales. References: (1) C. Chatterjee and J. N. Rai. Indian Phytopathol 28:309, 1973. (2) H. Komada. Rev. Plant Prot. Res. 8:114, 1975.

First report of Plectosphaerella cucumerina on greenhouse cultured wild rocket (Diplotaxis tenuifolia) in Italy.

During spring 2012, symptoms of an unusual leaf spot disease were observed in several commercial greenhouses near Salerno (southern Italy) on plants of Diplotaxis tenuifolia (cv Selvatica). The first symptoms on leaves of affected plants consisted of small (1 mm) black-brown spots of irregular shape, later coalescing into larger spots, 1 cm in diameter. Spots were surrounded by a yellow halo, and were mostly located on the foliar limb, rib, and petiole. Affected leaves were often distorted, appearing hook-like. The disease was severe under 75 to 90% RH, at air temperature of 20 to 26°C, and caused severe production losses on about 50 ha. Particularly, affected tissues rotted quickly after packaging and during transit and commercialization of processed rocket. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then placed on potato dextrose agar (PDA) medium, containing 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed producing a whitish-orange mycelium when incubated under 12 h/day of fluorescent light at 22°C. The isolates obtained were purified on PDA. On this medium, they produced hyaline elliptical and ovoid conidia, sometimes one-septate, measuring 4.5 to 9.2 × 1.7 to 3.5 (average 6.8 × 2.6) μm. Conidia were born on phialides, measuring 6.8 to 20.2 × 1.3 to 3.1 (average 16.5 × 2.1) μm. Such characteristics are typical of Plectosphaerella sp. (2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (3) and sequenced. BLAST analysis of the 519-bp segment showed a 98% similarity with the sequence of Plectosphaerella cucumerina (GenBank Accession No. AB469880). The nucleotide sequence has been assigned the GenBank Accession JX185769. To confirm pathogenicity, tests were conducted on 45-day-old D. tenuifolia plants. Plants (21/treatment), grown in 15 liter pots (7 plants/pot) were inoculated by spraying a 1 × 106 CFU/ml conidial suspension of one isolate of P. cucumerina, prepared from 10-day-old cultures, grown on PDA. Inoculated plants were maintained in a growth chamber at 23 ± 1°C, at 90% RH for 4 days. Non-inoculated plants served as control. Inoculated plants showed the typical first leaf spots 6 days after the artificial inoculation. Four days later, spots enlarged and leaves became distorted, showing chlorosis. Non-inoculated plants remained healthy. P. cucumerina was reisolated from inoculated plants. The pathogenicity test was conducted twice with identical results. This is, to our knowledge, the first report of P. cucumerina on D. tenuifolia in Italy as well as worldwide. P. cucumerina has been described as associated with root and collar rots of other horticultural crops in southern Italy (1). Due to the importance of the crop in Italy, this disease can cause serious economic losses. References: (1) A. Carlucci et al., Persoonia, 28:34, 2012. (2) M. E. Palm et al. Mycologia, 87:397, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.