HabiThreats toolkit | Publications

A new race of Fusarium oxysporum f. sp. lactucae of lettuce

Authors: Gilardi, G., Franco Ortega, S., Rijswick, P. C. J., Ortu, G., Gullino, M. L., & Garibaldi, A. (2017)

Fusarium oxysporum f. sp. lactucae, the causal agent of fusarium wilt of lettuce (Lactuca sativa), occurs in most countries in which lettuce is grown and causes serious economic losses. Three races (1, 2 and 3) of the pathogen have previously been identified on the basis of their ability to cause disease on differential lettuce cultivars, as well as by means of molecular tools developed to characterize different races of this pathogen. Only race 1 has been detected in Europe so far. In this study, two isolates of F. oxysporum, obtained from lettuce plants grown in the Netherlands showing symptoms of wilt, have been characterized by combining the study of pathogenicity with differential cultivars of lettuce and molecular assays to determine whether the isolates are different from the known races of F. oxysporum f. sp. lactucae. This study reports the presence of F. oxysporum f. sp. lactucae for the first time in the Netherlands. The causal pathogen has been identified, using the IRAP‐SCAR technique, as a new race of F. oxysporum f. sp. lactucae. Specific primers have been designed to identify this new race.

Contamination of moth mullein (Verbascum blattaria L.) seeds by Phoma novae-verbascicola

Authors: Bertetti, D., Ortu, G., Gullino, M. L., & Garibaldi, A. (2016)

Verbascum blattaria (Scrophulariaceae family) is a hardy perennial species that is used for the edges and flower beds of low-maintenance gardens. Phoma novae-verbascicola causes light brown necrotic spots on the leaves of V. blattaria seedlings. In order to demonstrate the seed transmission of this pathogen, several V. blattaria seeds belonging to three samples collected in 2013, were tested in vitro to detect the presence of P. novae- verbascicola. Two samples were found to be contaminated and colonies of the pathogen were isolated from the tested seeds. Phoma novae-verbascicola was identified from the morphological features observed in vitro and through an ITS (Internal Transcribed Spacer) analysis. The virulence of one isolate was confirmed by means of a pathogenicity test. This work demonstrates that P. novae-verbascicola can be transmitted by affected V. blattaria seeds.

Detection of mefenoxam-resistant strains of Peronospora belbahrii, the causal agent of basil downy mildew, transmitted through infected seeds

Authors: Pintore, I., Gilardi, G., Gullino, M. L., & Garibaldi, A. (2016)

Epidemics of basil downy mildew (DM) incited by Peronospora belbahrii have been very severe in Italy since 2013, in part due to the very favorable weather conditions, and losses have occurred in many commercial farms, even after repeated mefenoxam treatments. DM populations from basil plants and seeds, which are associated with failure in downy mildew control under field and greenhouse conditions, have been tested for their sensitivity to mefenoxam. Basil plants were inoculated with a sporangial suspension of seven DM populations and treated, before and after inoculation with the pathogen, with different dosages of mefenoxam: 100 μg/ml, which corresponds to the currently applied field dosage, 200 μg/ml and 1000 μg/ml. Azoxystrobin was used at the field dosage as the chemical control. Three out of four DM populations from seeds and two out of three from basil plants were found to be able to infect basil plants in the presence of 100 μg/ml and 200 μg/ml of mefenoxam, while the field dosage of azoxystrobin (186 μg/ml) was found to be completely effective. The sensitive populations of P. belbahrii were completely controlled by the field dosage of both chemicals also 14 days after the last treatment. This study provides new information on the potential risk of introducing mefenoxam-resistant P. belbahrii inoculum in the field through seeds infected by resistant strains.

Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds

Authors: Carneiro, G. A., Matić, S., Ortu, G., Garibaldi, A., Spadaro, D., & Gullino, M. L. (2017)

Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

Development of loop-mediated isothermal amplification assays for the detection of seedborne fungal pathogens, Fusarium fujikuroi and Magnaporthe oryzae, in rice seeds

Authors: Franco Ortega, S., Tomlinson, J., Hodgetts, J., Spadaro, D., GULLINO, L. M., & Boonham, N. (2018)

Bakanae disease (caused by Fusarium fujikuroi) and rice blast (caused by Magnaporthe oryzae) are two of the most important seedborne pathogens of rice. The detection of both pathogens in rice seed is necessary to maintain high quality standards and avoid production losses. Currently, blotter tests are used followed by morphological identification of the developing pathogens to provide an incidence of infection in seed lots. Two loop-mediated isothermal amplification assays were developed with primers designed to target the elongation factor 1-α sequence of F. fujikuroi and the calmodulin sequence of M. oryzae. The specificity, sensitivity, selectivity, repeatability, and reproducibility for each assay was assessed in line with the international validation standard published by the European and Mediterranean Plant Protection Organization (PM7/98). The results showed a limit of detection of 100 to 999 fg of DNA of F. fujikuroi and 10 to 99 pg of M. oryzae DNA. When combined with a commercial DNA extraction kit, the assays were demonstrated to be effective for use in detection of the pathogens in commercial batches of infected rice seed of different cultivars, giving results equivalent to the blotter method, thus demonstrating the reliability of the method for the surveillance of F. fujikuroi and M. oryzae in seed-testing laboratories.

Effect of a climate change scenario on Fusarium equiseti leaf spot on wild rocket and radish under phytotron simulation

Authors: Gullino, M. L., Gilardi, G., & Garibaldi, A. (2017)

This study was undertaken by simulating the effects of increasing the temperature and CO2 values on the incidence and severity of F. equiseti on wild rocket (Diplotaxis tenuifolia) and radish (Raphanus sativus), under phytotron conditions. Two sets of 3 trials were carried out in which eight different temperature and CO2 combinations were tested:1) 400–450 ppm CO2, 18–22 °C; 2) 800–850 ppm CO2, 18–22 °C; 3) 400–450 ppm CO2, 22–26 °C, 4) 800–850 ppm CO2, 22–26 °C, 5) 400–450 ppm CO2, 26–30 °C; 6) 800–850 ppm CO2, 26–30 °C; 7) 400–450 ppm CO2, 14–18 °C; 8) 800–850 ppm CO2, 14–18 °C. The temperature and CO2 levels were significant factors of influence on disease incidence (DI) and severity (DS) in all the trials, and their combination significantly influenced the DI and DS of F. equiseti leaf spot on both hosts. Disease incidence and severity increased on wild rocket at 850 ppm of CO2, in comparison to 450 ppm, in each tested temperature range. The highest CO2 value on radish, for all the tested temperature regimes, caused an increase in DI and DS, which resulted statistically significant at the highest tested temperature range. The results obtained in this study add more concern to the possible negative effects of the spread of F. equiseti on vegetables in Italy as well as in other areas suffering from increased temperatures as a consequence of climate changes.

Effect of different temperatures and CO2 levels on Alternaria toxins produced on cultivated rocket, cabbage and cauliflower

Authors: Siciliano, I., Berta, F., Bosio, P., Gullino, M. L., & Garibaldi, A. (2017)

Over the last 100 years, the global mean temperature has increased and has influenced several key factors that affect the occurrence and severity of fungal diseases. The effect of an increase in CO2 concentration and temperature on disease caused by four Alternaria strains and their mycotoxin production on cultivated rocket, cabbage and cauliflower plants has been investigated in this study. Six different temperature and CO2 combinations were considered: (1) 400-450 ppm CO2, 14-18 °C; (2) 800-850 ppm CO2, 14-18 °C; (3) 400-450 ppm CO2, 18-22 °C; (4) 800-850 ppm CO2, 18-22 °C; (5) 400-450 ppm CO2, 22-26 °C; and (6) 800-850 ppm CO2, 22-26 °C. Higher levels of CO2 and temperature have been found to significantly influence the disease index of the infected plants. In fact, the disease index was significantly increased at 22-26 °C and 800-850 ppm of CO2 for all the host plants. Tenuazonic acid (TeA), alternariol, alternariol monomethyl ether and tentoxin were analysed for each climate condition using HPLC-MS/MS, and disease severity was evaluated. Higher temperature influences environmental conditions and different factors involved in plant-pathogen interaction. Temperature was the main factor involved in disease severity, while host plants and strains were found to be the factors that had the most influence on the variation of the production of mycotoxins. A large variability in the production of mycotoxins among the different host plants was observed, but TeA was always the most frequently produced mycotoxin.

Effect of elevated atmospheric CO 2 and temperature on the chemical and biological control of powdery mildew of zucchini and the Phoma leaf spot of leaf beet

Authors: Gilardi, G., Gisi, U., Garibaldi, A., & Gullino, M. L. (2017)

The effects of increased temperature and CO2 levels on the effectiveness of four fungicides and one microbial treatment on disease development in zucchini-Podosphaera xanthii and leaf beet-Phoma betae pathosystems, kept under phytotron conditions, have been evaluated in this study. Six CO2 and temperature combinations have been tested for each pathosystem in four experimental trials. Penconazole and sulphur treatments, applied under a simulated CO2 and temperature increase scenario, have shown an efficacy in powdery mildew control that ranged from 85.0 to 88.9 % for penconazole and from 89.9 to 92.6 % for sulphur, and the treatments have therefore resulted to be equally effective compared to that observed under 400–450 ppm conditions. The disease control provided by A. quisqualis was significantly improved under an increased CO2 concentration of 800–850 ppm at 26–30 °C, by 23.3 % for disease incidence and 22.8 % for disease severity. The effectiveness of both mancozeb and azoxystrobin against Phoma leaf spot is affected by high levels of CO2. The efficacy of mancozeb and azoxystrobin has been improved significantly by 15.3 % and 20.6 %, respectively, under 800–850 ppm of CO2 and 22–26 °C, compared to the efficacy observed under standard conditions of CO2. More attention should be paid to the efficacy of chemical and biological control measures considering the predicted future climate changes.

Effect of elevated atmospheric CO2 and temperature on the disease severity of rocket plants caused by Fusarium wilt under phytotron conditions

Authors: Chitarra, W., Siciliano, I., Ferrocino, I., Gullino, M. L., & Garibaldi, A. (2015)

The severity of F. oxysporum f.sp. conglutinans on rocket plants grown under simulated climate change conditions has been studied. The rocket plants were cultivated on an infested substrate (4 log CFU g-1) and a non-infested substrate over three cycles. Pots were placed in six phytotrons in order to simulate different environmental conditions: 1) 400–450 ppm CO2, 18–22°C; 2) 800–850 ppm CO2, 18–22°C; 3) 400–450 ppm CO2, 22–26°C, 4) 800–850 ppm CO2, 22–26°C, 5) 400–450 ppm CO2, 26–30°C; 6) 800–850 ppm CO2, 26–30°C. Substrates from the infested and control samples were collected from each phytotron at 0, 60 and 120 days after transplanting. The disease index, microbial abundance, leaf physiological performances, root exudates and variability in the fungal profiles were monitored. The disease index was found to be significantly influenced by higher levels of temperature and CO2. Plate counts showed that fungal and bacterial development was not affected by the different CO2 and temperature levels, but a significant decreasing trend was observed from 0 up to 120 days. Conversely, the F. oxysporum f.sp. conglutinans plate counts did not show any significantly decrease from 0 up to 120 days. The fungal profiles, evaluated by means of polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), showed a relationship to temperature and CO2 on fungal diversity profiles. Different exudation patterns were observed when the controls and infested plants were compared, and it was found that both CO2 and temperature can influence the release of compounds from the roots of rocket plants. In short, the results show that global climate changes could influence disease incidence, probably through plant-mediated effects, caused by soilborne pathogens.

Emerging foliar and soil-borne pathogens of leafy vegetable crops: a possible threat to Europe

Authors: Gilardi, G., Gullino, M. L., & Garibaldi, A. (2018)

Italy is one of the leading countries for the production and consumption of ready-to-eat salads. This has led to a steady increase in the area used over the last decade for the growth of leafy vegetables under intensive systems. The recent new diseases that affect lettuce, wild and cultivated rocket, lamb's lettuce, spinach and basil are reported here: Plectosphaerella cucumerina on wild rocket, endive and lamb's lettuce, Fusarium equiseti on wild and cultivated rocket and lettuce, Myrothecium verrucaria on spinach and wild rocket, Myrothecium roridum on lamb's lettuce, Allophoma tropica on lettuce and Alternaria sp. on basil and rocket are among the new foliar pathogens in Italy. Among the soil-borne pathogens, Pythium aphanidermatum, Pythium irregulare and Pythium Cluster B2a have recently been isolated in Italy on spinach and Swiss chard, lamb's lettuce and lettuce, while Fusarium oxysporum f. sp. lactucae, which causes lettuce wilt, is gradually spreading to new countries. Some of the new pathogens have been found to be transmitted by seed and typical of tropical climate and are thus favoured by the increases in temperature linked to climate change. The globalization of markets, climate change and intensive cultivation are among the factors responsible for the proliferation and spread of some of these new pathogens that are ‘alien’ to Italian production systems.

Fast and specific detection of the invasive forest pathogen Heterobasidion irregulare through a Loop-mediated isothermal AMPlification (LAMP) assay

Authors: Sillo, F., Giordano, L., & Gonthier, P. (2018)

Heterobasidion irregulare is one of the most destructive fungal pathogens of pines in North America and was accidentally introduced into central Italy, where it has become invasive. The fungus is currently recommended for regulation by the European and Mediterranean Plant Protection Organisation (EPPO). In this work, an efficient diagnostic tool for the early detection of H. irregulare based on Loop‐mediated isothermal AMPlification (LAMP) coupled with two different DNA extraction methods was developed. The LAMP assay showed high specificity and good sensitivity, with a limit of detection of about 20 picograms of target DNA and time of detection of less than 40 min. The assay was successfully tested on a variety of different samples, including fungal fruiting bodies, infected plants and colonized wood. A survey on environmental samples collected in the field was also performed using the LAMP assay coupled with a rapid DNA extraction method. The possible applications of this molecular diagnostic tool encompass the monitoring of pine forests surrounding the current invasion area, laboratory or in‐field analyses of samples from suspected trees, and the surveillance in the ports of entry of wood imported from North America.

First Report of a Leaf Spot Caused by Boeremia exigua var. linicola on Autumn Sage (Salvia greggii) in Italy and Worldwide

Authors: Garibaldi, A., Gilardi, G., Franco Ortega, S., & Gullino, M. L. (2016)

Salvia greggii, autumn sage, is a perennial shrub, belonging to the Lamiaceae. Due to its pink to red flowers and its rusticity, this species is used in gardens in mixed borders and containers. During the fall of 2014, in a mountain garden located near Biella, northern Italy (45.6121660° N; 8.0562970° E; elev. 850 m), a leaf spot was observed on 12-month-old plants. All plants grown in the garden were affected, with 20 to 30% of leaves showing symptoms. First symptoms consisted of circular necrotic spots, with diameter ranging between 10 and 70 mm, well defined by a brown margin, and generally the center of the necrosis was bleached. Severely infected leaves became chlorotic and abscised. Infected plants rarely died, but the presence of lesions on mature plants decreased their aesthetic quality. The disease started from basal leaves on plants grown in shade and at higher relative humidity, progressing up and outward on the plants, until they were almost completely defoliated. A fungus was consistently isolated from the margin of the necrotic lesions using potato dextrose agar amended with 25 mg/liter streptomycin sulfate. After 7 days, black 140 to 225-μm-diameter pycnidia developed, releasing hyaline, elliptical, and aseptate conidia measuring 4.8 to 9.2 × 1.6 to 4.3 (avg. 6.9 × 3.1) μm.

First Report of Botrytis Blight Caused by Botrytis cinerea on Anemone japonica in Italy

Authors: Garibaldi, A., Gilardi, G., Ortega, S. F., & Gullino, M. L. (2016)

Anemone japonica (Japanese anemone or windflower) is an ornamental, late summer-flowering perennial originating from China. It is resistant to drought and low temperatures. In September 2014, symptoms of a previously unknown blight were observed on 2- to 5-year-old plants in a private garden near Biella, northern Italy (45°36′00″ N; 8°03′00″ E; 850 m). Affected leaves showed first chlorotic then necrotic spots that enlarged, became irregular, water-soaked and brown, often with petal dropping. Infected petals withered and turned tannish brown, often sticking together. Severely infected tissues eventually became completely rotted, and later desiccated. Infection also spread through the leaf petiole. A soft and gray mycelium was observed on symptomatic tissues. The disease affected ∼10 to 25% of plants in late August-early September, and 40 to 60% in October (10 and 15°C). Generally, in the garden infected flower parts served as an inoculum source for leaf infection. Diseased tissue was excised from affected leaves, immersed in 1% sodium hypochlorite for 10 s, rinsed in sterile water, then cultured on potato dextrose agar (PDA) medium amended with 25 mg/liter of streptomycin sulfate, and incubated in 12-h photoperiod at 22 ± 1°C. Abundant mycelium developed with branched conidiophores with enlarged apical cells in 10-day-old colonies. Conidia were smooth, ovoid to elongate light ash-colored, unicellular, ovoid, 11.4 to 19.3 × 10.3 to 11.6 (avg. 14.5 × 11.6) µm. Sclerotia, were flat, irregularly shaped and black, 1.5 to 4.8 × 1.1 to 3.2 mm.

First Report of Fusarium oxysporum f. sp. radicis-cucumerinum causing wilt on Cucumber (Cucumis sativus L.) in Italy

Authors: Garibaldi, A., Gilardi, G., Ortu, G., & Gullino, M. L. (2016)

During the spring of 2015, plants of lamb’s lettuce (Valerianella olitoria) ‘Palace’ grown under plastic-house in the Lombardy Region (Northern Italy) showed symptoms of a previously unknown foliar disease. Symptoms were observed on 15-day-old plants and consisted of small, circular, gray-brown leaf spots,1 to 3 mm in diameter, with a well defined border. As the lesions expanded (up to 30 mm in diameter), they developed concentric rings, coalesced, and the leaf blighted down. Slightly green to black sporodochia appeared on the affected leaves, at different age. Roots were asymptomatic. Fifteen hectares of plastic-houses were affected with 5 to 15% of plants exhibiting symptoms. Isolations were carried out from symptomatic affected leaf tissues sections (each 1 mm2) dipped in 1% sodium hypochlorite for 1 min and rinsed in sterilized water, then placed on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. A white slow-growing fungus was obtained and, 7 days after isolation, developed black sporodochia similar to those present on the affected leaves. Conidia were cylindrical, non-septate, 3.1 to 7.2 × 1.5 to 3.3 (avg. 5.4 × 2.4) µm. The morphology corresponded to Myrothecium roridum Tode ex Fr. (Fitton and Holliday 1970).

First Report of Leaf Spot Caused by Colletotrichum fioriniae on Mexican Bush Sage (Salvia leucantha) in Italy

Authors: Garibaldi, A., Gilardi, G., Franco-Ortega, S., & Gullino, M. L. (2016)

Salvia leucantha, common name Mexican bush sage, is an herbaceous perennial belonging to the Lamiaceae family. Native to subtropical and tropical conifer forests in central and eastern Mexico, this species is used in gardens in mixed borders at warm latitudes due to its white flowers emerging from colored bracts and its rusticity. The plant is also used for the production of essential oils. During the summer-fall of 2014, in a mountain garden located near Biella, northern Italy (45.6121660° N; 8.0562970° E; 850 m elevation), a previously unreported leaf spot was observed on 6- to 8-month-old plants. At temperatures between 15 and 25°C, 80% of all Mexican Bush Sage plants grown in the garden were affected. The first symptoms consisted of small necrotic spots, measuring 10 to 30 mm, that increased in number to cover a large percentage of the leaf, which eventually collapsed. The disease started from basal leaves on plants grown in shade and at higher RH. Severely diseased plants were defoliated 30 to 35 days after the first symptoms appeared. Several isolations were done from infected tissues on potato dextrose agar amended with 25 mg/liter of streptomycin sulfate over six months. A Colletotrichum sp. was consistently recovered from the symptomatic tissues (Bailey and Jeger 1992). Hyaline, elliptical to cylindrical, aseptate and thin-walled conidia (10.7 to 17.1 × 4.6 to 6.9, average 14.6 × 5.6 μm) were abundantly produced in acervuli (85 to 197, 136 μm average) produced in gray mycelium. Genomic DNA was extracted with E.Z.N.A. Plant DNA Kit (Omega Bio-Tek) from 7-day-old pure culture.

First report of leaf spot caused by Colletotrichum kahawae on cultivated rocket (Eruca sativa) in Italy

Authors: Garibaldi, A., Gilardi, G., Puglisi, I., Cacciola, S. O., & Gullino, M. L. (2016)

Eruca sativa common name cultivated rocket, is an annual species in the Brassicaceae, grown for fresh consumption. In fall 2014, in a commercial plastic-house in northern Italy, where the crop was grown for 7 years in the same soil, a new leaf spot was observed on rocket plants (‘Coltivata’) 7 to 35 days after sowing. The incidence ranged from 5 to 15% in the more humid area of the tunnel, with 5 to 75% of the affected leaf area showing white spots. Symptoms first appeared on the tip or edge of leaf as pale white or cream-colored circular spots 5 to 50 mm in diameter. Spots had thin dark brown or black borders. They enlarged, with round or irregular shapes, to form extensive dead areas. The disease developed at 20 to 28°C and 75 to 90% relative humidity. Isolations were carried out from affected leaf tissues (1 mm2) dipped in 1% sodium hypochlorite for 1 min, then rinsed in sterilized water, and placed onto PDA amended with 25 mg/liter of streptomycin sulfate. A Colletotrichum sp. (Bailey and Jeger 1992) was consistently recovered. Hyaline, cylindrical, aseptate, and thin-walled conidia (9.3 to 16.5 × 3.4 to 6.3 μm, avg. 14.4 × 4.7 μm; n = 41) were produced abundantly in acervuli (79.0 to 91.5 µm; n = 15) in gray mycelium. Genomic DNA was extracted from 10 mg of fresh mycelium collected from PDA plates of one representative, single-conidium isolate. The ITS1-5.8S-ITS2 region of ribosomal DNA (rDNA) and a fragment of the beta-tubulin 2 gene (TUB2) between exons 2 and 6 were amplified using 200 ng of genomic DNA as template.

18. First Report of Leaf Spot of Campanula medium Caused by Alternaria alternata in Italy

Authors: Garibaldi, A., Ortega, S. F., Gilardi, G., & Gullino, M. L. (2016)

Campanula medium L. (Canterbury bell, also known as bell flower) is an annual or biennial low maintenance flower plant belonging to the Campanulaceae, typically grown in parks and gardens. During summer 2015, extensive necrosis was observed on leaves of plants grown in a private garden near Biella in northern Italy (45.612166° N, 8.056297° E) at an altitude of 850 m. The garden, about 8,000 m2, was designed a century ago, and it is planted with many other native acid-loving species. The disease affected 30 to 70% of about 100 plants (10 to 20 months old), grown under high relative humidity, at temperatures of 15 to 26°C. The first symptoms were usually brown to black, circular lesions surrounded by a yellow halo, frequently localized on the tips and margins of leaves. Lesions sometimes coalesced and infected leaves eventually turned brown. In some cases, stems were affected, and plants died; no symptoms were observed on flowers since they were not present at the appearance of symptoms. A fungus was consistently isolated from infected leaves on potato dextrose agar (PDA), added with streptomycin sulfate at 25 mg/liter. The monoconidial isolate AltCm42, grown for 7 days with a 12-h photoperiod at 20 to 22°C on PDA, produced a greenish mycelium with dark brown conidia, in chains of 2 to 8 elements, ovoid, elliptical or obclavate in shape, with a beak. Conidia showed 2 to 6 transverse and 0 to 4 longitudinal septa, and measured 17.7 to 48.4 (avg. 24.4) × 6.8 to 8.9 (avg. 7.8) µm. The beaks showed a color generally lighter than the rest of the conidia, 2.2 to 9.4 (average 6.2) µm long. Based on its morphological characteristics, the pathogen was identified as Alternaria sp. (Simmons 2007). Genomic DNA was extracted with E.Z.N.A.

First report of leaf spot of lamb’s lettuce (Valerianella olitoria) caused by Myrothecium roridum in Italy

Authors: Garibaldi, A., Gilardi, G., Franco-Ortega, S., & Gullino, M. L. (2016)

During the spring of 2015, plants of lamb’s lettuce (Valerianella olitoria) ‘Palace’ grown under plastic-house in the Lombardy Region (Northern Italy) showed symptoms of a previously unknown foliar disease. Symptoms were observed on 15-day-old plants and consisted of small, circular, gray-brown leaf spots,1 to 3 mm in diameter, with a well defined border. As the lesions expanded (up to 30 mm in diameter), they developed concentric rings, coalesced, and the leaf blighted down. Slightly green to black sporodochia appeared on the affected leaves, at different age. Roots were asymptomatic. Fifteen hectares of plastic-houses were affected with 5 to 15% of plants exhibiting symptoms. Isolations were carried out from symptomatic affected leaf tissues sections (each 1 mm2) dipped in 1% sodium hypochlorite for 1 min and rinsed in sterilized water, then placed on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. A white slow-growing fungus was obtained and, 7 days after isolation, developed black sporodochia similar to those present on the affected leaves. Conidia were cylindrical, non-septate, 3.1 to 7.2 × 1.5 to 3.3 (avg. 5.4 × 2.4) µm. The morphology corresponded to Myrothecium roridum Tode ex Fr. (Fitton and Holliday 1970). The ITS region of rDNA was amplified using the primers ITS1/ITS4, and sequenced. BLAST analysis (Altschul et al. 1997) of the 531-bp segment showed 99% similarity with M. roridum (GenBank Accession Nos. KF494828.1, KJ813720.1, and HQ115647.1). The nucleotide sequence has been assigned the GenBank Accession No. KT354921.

First Report of Leaf Spot of Spinach (Spinacia oleracea) Caused by Myrothecium verrucaria in Italy

Authors: Garibaldi, A., Gilardi, G., Franco-Ortega, S., & Gullino, M. L. (2016)

During the spring of 2015, plants of spinach (Spinacia oleracea L.) ‘Donkey’ were grown using conventional control strategies in plastic houses in the Lumbardy Region near Brescia (Northern Italy). The plants showed symptoms of a previously unknown foliar disease on 15- to 20-day-old plants that consisted of small, circular, sunken, gray-brown spots (1 to 2 mm in diameter) with a well-defined border. As the lesions expanded (up to 30 mm in diameter) they developed concentric rings, coalesced, and then the affected tissues became water-soaked. Creamy to black sporodochia appeared on the affected leaves. Roots were asymptomatic. Five hectares of plastic houses irrigated with well water by overhead sprinklers were affected, with 5 to 10% of plants exhibiting symptoms. Affected plants showed 30 to 60% incidence of diseased leaves. Isolations were carried out on symptomatic leaf-tissue sections (each 1 mm2) dipped in 1% sodium hypochlorite for 1 min, rinsed in sterilized water, dried on sterilized filter paper, and then placed on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. A white slow-growing fungus was obtained that developed black sporodochia 7 to 10 days after plating, similar to those present on the affected leaves. Conidia were cylindrical, nonseptate, without appendages, measuring 5.8 to 9.2 and 1.9 to 2.8 (avg. of 30 conidia, 7.6 ± 1.14 × 2.4 ± 0.22) µm. The morphological characteristics of the isolated pathogen corresponded to those of Myrothecium spp. (Domsch et al. 1980). The Internal Transcribed Spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (Altschul et al. 1997) of the 516-bp segment showed a 100% similarity with M. verrucaria KR708633.1. A GenBank Accession No. KT354922 was assigned.

Identification of Fusarium Oxysporum f. Sp. Opuntiarum on new hosts of the Cactaceae and Euphorbiaceae families

Authors: Bertetti, D., Ortu, G., Gullino, M. L., & Garibaldi, A. (2017)

Fusarium oxysporum has recently been detected in commercial nurseries in the Ligurian region (northern Italy) on new succulent plants belonging to the Cactaceae family (Astrophytum myriostigma, Cereus marginatus var. cristata, C. peruvianus monstruosus and C. peruvianus florida) and to the Euphorbiaceae family (Euphorbia mammillaris). The pathogen has been identified, for all the new hosts, from morphological characteristics observed in vitro. The identifications have been confirmed by means of ITS (Internal Transcribed Spacer) analysis and/or by Translation Elongation Factor 1α (TEF) analysis. The aim of this work was to identify the forma specialis of the F. oxysporum isolates obtained from new succulent plants. This has been investigated by means of phylogenetic analysis based on the TEF gene and intergenic spacer (IGS), carried out on single-spore isolates, together with pathogenicity assays. The results of this research led to include four new succulent hosts from Cactaceae as additional hosts to F. oxysporum f. sp. opuntiarum. This forma specialis has been identified for the first time on a new host (Euphorbia mammillaris) not belonging to the Cactaceae family.

Nursery treatments with non-conventional products against crown and root rot, caused by Phytophthora capsici, on zucchini

Authors: Gilardi, G., Pintore, I., Gullino, M. L., & Garibaldi, A. (2017)

Phytophthora capsici, a pathogen causing crown and root rot of zucchini in southern Italy since the 1980s, has recently been observed in open field in northern Italy, causing severe losses. Since chemical control on zucchini is complicated by a limited availability of registered chemicals, as well as by the scalar harvest, a number of resistance inducers, organic amendments, biocontrol agents and fungicides were tested against P. capsici, under greenhouse conditions. Experiments were carried out at the nursery level, with different timing and number of applications. In the presence of a very high disease pressure, the best disease control was provided by mefenoxam, followed by the phosphite-based products, which acted as resistance inducers and also provided a positive effect on plant biomass. Acibenzolar-S-methyl, although effective, was sometimes phytotoxic. The biocontrol agents tested as well as the patented formulation of Brassica carinata defatted seed meals were not effective, providing results statistically similar to the untreated control. The efficacy of resistance inducers is interesting in view of their possible use in alternation with chemicals, or as stand-alone treatments in cultivation systems which do not adopt chemical control.

Potential for re-emergence of wheat stem rust in the United Kingdom

Authors: Lewis C.M., Persoons A., Bebber D.P., Kigathi R.N., Maintz J., Findlay K., Bueno-Sancho V., Corredor-Moreno P., Harrington S.A., Kangara N., Berlin A., García R., Germán S.E., Hanzalová A., Hodson D.P., Hovmøller M.S., Huerta-Espino J., Imtiaz M., Mirza J.I., Justesen A.F., Niks R.E., Omrani A., Patpour M., Pretorius Z.A., Roohparvar R., Sela H., Singh R.P., Steffenson B., Visser B., Fenwick P.M., Thomas J., Wulff B.B.H., Saunders D.G.O. (2018)

Wheat stem rust, a devastating disease of wheat and barley caused by the fungal pathogen Puccinia graminis f. sp. tritici, was largely eradicated in Western Europe during the mid-to-late twentieth century. However, isolated outbreaks have occurred in recent years. Here we investigate whether a lack of resistance in modern European varieties, increased presence of its alternate host barberry and changes in climatic conditions could be facilitating its resurgence. We report the first wheat stem rust occurrence in the United Kingdom in nearly 60 years, with only 20% of UK wheat varieties resistant to this strain. Climate changes over the past 25 years also suggest increasingly conducive conditions for infection. Furthermore, we document the first occurrence in decades of P. graminis on barberry in the UK . Our data illustrate that wheat stem rust does occur in the UK and, when climatic conditions are conducive, could severely harm wheat and barley production.

Rapid detection of Fusarium oxysporum f. sp. lactucae on soil, lettuce seeds and plants using loop‐mediated isothermal amplification

Authors: Franco Ortega, S., Tomlinson, J., Gilardi, G., Spadaro, D., Gullino, M. L., Garibaldi, A., & Boonham, N. (2018)

Fusarium oxysporum f. sp. lactucae (FOL) is a soil‐ and seedborne pathogen and the causal agent of fusarium wilt on lettuce. Four races have been identified within FOL, with different worldwide distribution. Several molecular techniques have been used to detect and identify this pathogen; however, not all of them have the optimal characteristics in terms of sensitivity to perform FOL detection in plant and seed material. A loop‐mediated isothermal amplification (LAMP) assay was developed based on the sequence‐characterized amplified region (SCAR) obtained in a previous rapid amplification of polymorphic DNA (RAPD) study. The LAMP assay has been validated according to the EPPO standard PM7/98. The LAMP assay was tested with lettuce seeds, soil and plant material, and can be used successfully to amplify DNA from each of these matrices. In seed lots artificially inoculated with FOL, the detection limit of the LAMP test was 0.004% infected seed.

Sexual Reproduction in the Fungal Foliar Pathogen Zymoseptoria tritici Is Driven by Antagonistic Density Dependence Mechanisms

Authors: Suffert, F., Delestre, G., & Gélisse, S. (2018)

This study provides empirical evidence for antagonistic density dependence mechanisms driving sexual reproduction in the wheat fungal pathogen Zymoseptoria tritici. Biparental crosses with 12 increasing inoculum concentrations, in controlled conditions, showed that sexual reproduction in Z. tritici was impacted by an Allee effect due to mate limitation and a competition with asexual multiplication for resource allocation. The highest number of ascospores discharged was reached at intermediate inoculum concentrations (from 5 × 104 conidia mL−1 to 106 conidia mL−1). Consistent with these results for controlled co-inoculation, we found that the intensity of sexual reproduction varied with both cropping period and the vertical position of the host tissues in the field, with a maximum between 25 and 35 cm above the ground. An optimal lesion density (disease severity of 30 to 45%) maximizing offspring (ascospores) number was established, and its eco-evolutionary consequences are considered here. Two ecological mechanisms may be involved: competition for resources between the two modes of reproduction (decrease in the host resources available for sexual reproduction due to their prior use in asexual multiplication), and competitive disequilibrium between the two parental isolates, due to differential interaction dynamics with the host, for example, leading to an imbalance between mating types. A conceptual model based on these results suggests that sexual reproduction plays a key role in the evolution of pathogenicity traits, including virulence and aggressiveness. Ecological knowledge about the determinants of sexual reproduction in Z. tritici may, therefore, open up new perspectives for the management of other fungal foliar pathogens with dual modes of reproduction.

Simulated elevated atmospheric CO2 and temperature affect the severity of bean and pelargonium rust

Authors: Gilardi, G., Puglliese, M., Gullino, M. L., & Garibaldi, A. (2016)

Experimental trials were carried out on bean (Phaseolus vulgaris L.)/Uromyces appendiculatus F. Strauss and pelargonium [Pelargonium zonale (L.) L’Hér.ex Aiton]/ Puccinia pelargonii-zonalis Doidge pathosystems, under phytotron conditions, to evaluate the effects of simulated elevated atmospheric CO2 concentrations, ranging from 800 to 850 ppm, compared with standard CO2, ranging from 400 to 450 ppm, and temperatures, ranging from 14 to 18, 18 to 22, 22 to 26 and 26 to 30 °C. A total of eight CO2 and temperature combinations were tested to establish their effects on the development of bean and pelargonium rusts. A doubled concentration of CO2, at the same temperature regime, was found to lead to an increase in U. appendiculatus disease severity on the beans. The combined CO2 and temperature factors significantly influenced the severity (p = 0.049) of the rust caused by U. appendiculatus. Moreover, Puccinia pelargonii–zonalis was observed to be more severe on pelargonium at the lowest tested temperatures, that is, between 14 and 22 °C. A high CO2 regime was shown to significantly increase disease severity at such temperatures. At the highest tested temperatures, that is, between 26 and 30 °C, which are generally not favourable for rust development, the increase in CO2 had no significant effect on disease severity.

Temperature and leaf wetness affect the severity of leaf spot on lettuce and wild rocket incited by Fusarium equiseti

Authors: Garibaldi, A., Gilardi, G., Berta, F., & Gullino, M. L. (2016)

Eight experimental trials have been carried out under controlled conditions in order to have a better understanding of the effect of temperature and leaf wetness duration on the incidence and severity of leaf spot caused by Fusarium equiseti on lettuce cv. Elisa and wild rocket cv. Grazia. Lettuce resulted very susceptible to F. equiseti, particularly at temperatures of 25–30 °C, with a higher disease index than 50% and a higher disease severity than 25%. At such temperatures, 1–3 h of leaf wetness were sufficient to cause a high disease incidence and severity, while at least 12 h of leaf wetness were necessary to cause high losses at lower temperatures. Disease incidence and severity were higher on the wild rocket at the highest temperatures (30–35 °C). Only one hour of leaf wetness was sufficient to cause significant levels of disease incidence and severity at the highest temperatures, while longer periods (6–12 h) were necessary to cause significant losses at lower temperatures. The possible causes of the recent spread of F. equiseti in northern Italy on a number of crops as well as the threat represented by such a pathogen are discussed hereafter.

Verrucarin A and roridin E produced on rocket by Myrothecium roridum under different temperatures and CO2 levels

Authors: Bosio, P., Siciliano, I., Gilardi, G., Gullino, M. L., & Garibaldi, A. (2017)

The behaviour of Myrothecium roridum, artificially inoculated on cultivated rocket (Eruca sativa), has been evaluated under eight different temperature and CO2 concentration combinations (from 14-18 °C to 26-30 °C and with 400-450 or 800-850 ppm of CO2). The pathogen isolate used for this study was inoculated on rocket and disease severity increased with high temperatures for both CO2 levels. Verrucarin A and roridin E mycotoxins were produced under all the tested temperatures at high CO2 conditions. The maximum level of verrucarin A was found at 14-18 °C and 800-850 ppm of CO2, and the maximum roridin E production was detected at 26-30 °C with 800-850 ppm of CO2. The results obtained in this study show that both the CO2 concentration and the temperature influence disease severity and mycotoxin production in different ways. An increase in temperature, which is favourable for attacks of the pathogen, could induce the spread of M. roridum in temperate regions, and this pathogen could take on even greater importance in the future, considering its ability to produce mycotoxins.

Verrucarin A and roridin E produced on spinach by Myrothecium verrucaria under different temperatures and CO 2 levels

Authors: Siciliano, I., Bosio, P., Gilardi, G., Gullino, M. L., & Garibaldi, A. (2017)

The behavior of Myrothecium verrucaria, artificially inoculated on spinach, was studied under seven different temperature conditions (from 5 to 35 °C) and under eight different combinations of temperature and CO2 concentration (14–30 °C and 775–870 or 1550–1650 mg/m3). The isolate used for this study was growing well on spinach, and the mycotoxins verrucarin A and roridin E were produced under all tested temperature and CO2 conditions. The maximum levels of verrucarin A (18.59 ng/g) and roridin E (49.62 ng/g) were found at a temperature of 26–30 °C and a CO2 level of 1550–1650 mg/m3. Rises in temperature as well as in temperature and CO2 concentrations had a significant effect by increasing Myrothecium leaf spots on spinach. The biosynthesis of verrucarin A was significantly increased at the highest temperature (35 °C), while roridin E was influenced by the CO2 concentration. These results show that a positive correlation between climate condition and macrocyclic trichothecene production is possible. However, because of the ability of M. verrucaria to produce mycotoxins, an increase in temperature could induce the spread of M. verrucaria in temperate regions; this pathogen may gain importance in the future.