The Impact of Climate Change on Vegetable Crop Diseases and Their Management: The Value of Phytotron Studies for the Agricultural Industry and Associated Stakeholders.

Climate change is having a significant impact on global agriculture, particularly on vegetable crops, which play a critical role in global nutrition. Recently, increasing research has concentrated on the impact of climate change on vegetable crop diseases, with several studies being conducted in phytotrons, which have been used to explore the effects of increased temperatures and CO2 concentrations to simulate future scenarios. This review focuses on the combined effects of temperature and carbon dioxide increases on foliar and soilborne vegetable diseases, as evaluated under phytotron conditions. The influence of climate change on mycotoxin production and disease management strategies is also explored through case studies. The results offer valuable information that can be used to guide both seed and agrochemical industries, as well as to develop disease-resistant varieties and innovative control measures, including biocontrol agents, considering the diseases that are likely to become prevalent under future climatic scenarios. Recommendations on how to manage vegetable diseases under ongoing climate change are proposed to facilitate plants' adaptation to and enhanced against the changing conditions. A proactive and comprehensive response to climate-induced challenges in vegetable farming is imperative to ensure food security and sustainability.

First Report of Gnomoniopsis fragrariae causing leaf spots on strawberry in Italy.

Strawberry (Fragaria × ananassa Duch.) is widely cultivated in Italy. During May-June 2022, mild symptoms of an unknown leaf spot disease appeared on 5-10% of June-bearing strawberry (cv. Elodì) plants transplanted in July 2021 in a commercial farm located in the province of Cuneo, North Italy. During September-November 2022, the symptoms appeared also on 10-15% of the plants transplanted in July 2022. The disease was scattered throughout the field, large 600 m2, both on new and senescent leaves. Fungicides (sulphur, Tiovit Jet; penconazole, Topas 10 EC) were applied to the plants according to integrated pest management during the growing period. The disease symptoms were purplish to brown necrotic leaf spots up to 1-3 mm in diameter and chlorotic leaf margins. Black lesions were occasionally observed on the petioles, appearing as small necrotic or larger elongated lesions causing leaf death. Peritechia were observed in planta after about 4 months from sampling and measured (144 to 239 µm and 200 to 291 µm, n = 10). Diseased leaves and petioles from about 10 plants were surface disinfested for 1 min in 1% NaClO, rinsed with sterile water and plated on potato dextrose agar (PDA) amended with 25 mg streptomycin sulphate/liter. A fungus with white cottony colonies was repeatedly recovered and maintained in pure culture on PDA. Biguttulate conidia with rounded ends were measured (4.3 to 8.0 µm and 1.2 to 2.9 µm, average 6.1× 2.3 µm, n = 50) from 21-day old colonies grown in PDA at 22°C and 12 h photoperiod. According to colony and conidia morphology, the isolate was identified as Gnomoniopsis sp. (Walker et al., 2010). The fungal DNA was extracted from a pure culture of one isolate selected as a representative (code FR2-22), by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). The identification was carried out by amplifying and sequencing the internal transcribed spacer (ITS) region and the partial translation elongation factor 1-α (TEF) gene using the primers ITS1/ITS4 and EF-728F/EF2 (Udayanga et al., 2021), respectively. The purified PCR products were sequenced at the BMR Genomics Centre (Padova, Italy) obtaining 551bp (ITS) and 652bp (TEF) sequences deposited in GenBank (Accession nos. OQ179950 and OQ190173, respectively). A BLASTn search of both sequences revealed to be 100% identical to the ITS and TEF loci of Gnomoniopsis fructicola sequences of the isolates VPRI_15547 and CBS 275.51 deposited in GenBank with accession Nos. MT378345 and MT383092. The pathogenicity of the isolate FR2-22 was assessed in two trials by biological tests (3 replicates with 1 plant per replicate/pot) in two greenhouse compartments, kept at temperature 20-24°C and at humidity 80-90%. Healthy leaves of forty-day-old strawberry plants (cv. Elodì) were sprayed with 1-5 x106 conidia/ml obtained from the FR2-22 isolate grown on PDA at 25°C for 20 days. The control (water-sprayed plants) was kept in the same conditions. Small leaf spots similar to the symptoms previously observed in the farm were observed 15 days post inoculation. Furthermore, 30 to 40% of leaves developed symptoms similar to those observed in the field after 25-40 days, while the control remained health. The same fungal isolate was repeatedly reisolated from the affected leaves and petioles and identified based on TEF sequencing. Gnomoniopsis fragariae comb. nov., designed as new name for Gnomoniopsis fructicola (Udayanga et al., 2021), has previously been reported on Fragaria × ananassa plants in Australia and in the USA (Farr and Rossman, 2023). To the best of our knowledge, this is the first report of G. fragariae on strawberry in Italy. The impact of the disease caused by this pathogen could be of high importance in the future of strawberry production in Italy. Healthy propagation material and strict disease management practices in nurseries is a requirement to avoid disease epidemics.

Microbial-Based Products to Control Soil-Borne Pathogens: Methods to Improve Efficacy and to Assess Impacts on Microbiome.

Microbial-based products (either as biopesticide or biofertilizers) have a long history of application, though their use is still limited, mainly due to a perceived low and inconsistent efficacy under field conditions. However, their efficacy has always been compared to chemical products, which have a completely different mechanism of action and production process, following the chemical paradigm of agricultural production. This paradigm has also been applied to regulatory processes, particularly for biopesticides, making the marketing of microbial-based formulations difficult. Increased knowledge about bioinocula behavior after application to the soil and their impact on soil microbiome should foster better exploitation of microbial-based products in a complex environment such as the soil. Moreover, the multifunctional capacity of microbial strains with regard to plant growth promotion and protection should also be considered in this respect. Therefore, the methods utilized for these studies are key to improving the knowledge and understanding of microbial-based product activity and improving their efficacy, which, from farmers' point of view, is the parameter to assess the usefulness of a treatment. In this review, we are thus addressing aspects related to the production and formulation process, highlighting the methods that can be used to evaluate the functioning and impact of microbial-based products on soil microbiome, as tools supporting their use and marketing.

Biological and molecular interplay between two viruses and powdery and downy mildews in two grapevine cultivars.

Grapevine may be affected simultaneously by several pathogens whose complex interplay is largely unknown. We studied the effects of infection by two grapevine viruses on powdery mildew and downy mildew development and the molecular modifications induced in grapevines by their multiple interactions. Grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) were transmitted by in vitro-grafting to Vitis vinifera cv Nebbiolo and Chardonnay virus-free plantlets regenerated by somatic embryogenesis. Grapevines were then artificially inoculated in the greenhouse with either Plasmopara viticola or Erysiphe necator spores. GFLV-infected plants showed a reduction in severity of the diseases caused by powdery and downy mildews in comparison to virus-free plants. GFLV induced the overexpression of stilbene synthase genes, pathogenesis-related proteins, and influenced the genes involved in carbohydrate metabolism in grapevine. These transcriptional changes suggest improved innate plant immunity, which makes the GFLV-infected grapevines less susceptible to other biotic attacks. This, however, cannot be extrapolated to GRSPaV as it was unable to promote protection against the fungal/oomycete pathogens. In these multiple interactions, the grapevine genotype seemed to have a crucial role: in 'Nebbiolo', the virus-induced molecular changes were different from those observed in 'Chardonnay', suggesting that different metabolic pathways may be involved in protection against fungal/oomycete pathogens. These results indicate that complex interactions do exist between grapevine and its different pathogens and represent the first study on a topic that still is largely unexplored.

Characterization of Colletotrichum ocimi Population Associated with Black Spot of Sweet Basil (Ocimum basilicum) in Northern Italy.

Black spot is a major foliar disease of sweet basil (Ocimum basilicum) present in a typical cultivation area of northern Italy, including the Liguria and southern Piedmont regions, where this aromatic herb is an economically important crop. In this study, 15 Colletotrichum isolates obtained from sweet basil plants with symptoms of black spot sampled in this area were characterized morphologically and by nuclear DNA analysis using internal transcribed spacers (ITS) and intervening 5.8S nrDNA as well as part of the ß-tubulin gene (TUB2) regions as barcode markers. Analysis revealed all but one isolate belonged to the recently described species C. ocimi of the C. destructivum species complex. Only one isolate was identified as C. destructivum sensu stricto (s.s.). In pathogenicity tests on sweet basil, both C. ocimi and C. destructivum s.s. isolates incited typical symptoms of black spot, showing that although C. ocimi prevails in this basil production area, it is not the sole causal agent of black spot in northern Italy. While no other hosts of C. ocimi are known worldwide, the close related species C. destructivum has a broad host range, suggesting a speciation process of C. ocimi within this species complex driven by adaptation to the host.

Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae.

BACKGROUND: Cytochrome P450 enzymes (CYPs) are monooxygenases present in every domain of life. In fungi CYPs are involved in virulence. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae, is the most serious disease of lettuce. F. oxysporum f.sp. lactucae MSA35 is an antagonistic fungus. Pathogenic formae specialis of F. oxysporum possess a CYP belonging to the new family CYP505. This enzyme hydroxylates saturated fatty acids that play a role in plant defence. METHODS: Molecular tools were adopted to search for cyp505 gene in MSA35 genome. cyp505 gene expression analysis in pathogenic and antagonistic Fusarium was performed. The enzyme was expressed in its recombinant form and used for catalytic reactions with fatty acids, the products of which were characterized by mass spectrometry analysis. RESULTS: A novel MSA35 self-sufficient CYP505 is differentially expressed in antagonistic and pathogenic F. oxysporum. Its expression is induced by the host plant lettuce in both pathogenesis and antagonism during the early phase of the interaction, while it is silenced during the late phase only in antagonistic Fusarium. Mass-spectrometry investigations proved that CYP505A1 mono-hydroxylates lauric, palmitic and stearic acids. CONCLUSIONS: The ability of CYP505A1 to oxidize fatty acids present in the cortical cell membranes together with its differential expression in its Fusarium antagonistic form point out to the possibility that this enzyme is associated with Fusarium pathogenicity in lettuce. GENERAL SIGNIFICANCE: The CYP505 clan is present in pathogenic fungal phyla, making CYP505A1 enzyme a putative candidate as a new target for the development of novel antifungal molecules.

Ready-to-Eat Salad Crops: A Plant Pathogen's Heaven.

The ready-to-eat salad sector, also called fresh-cut or bagged salads, is a fast-growing segment of the fresh-food industry. The dynamism and specialization of this sector, together with the lack of adequate crop rotation, the globalization of the seed market, and climate change, are the main causes of the development of many new diseases that cause severe production losses. Newly detected diseases of the most important crops grown (lettuce, wild and cultivated rocket, lamb's lettuce, chicory, endive, basil, spinach, and Swiss chard) are critically discussed. The management of these diseases represents a formidable challenge, since few fungicides are registered on these minor-use crops. An interesting feature of the ready-to-eat salad sector is that most crops are grown under protection, often in soilless systems, which provide an environment helpful to the implementation of innovative control methods. Current trends in disease management are discussed, with special focus on the most sustainable practices.

Emergence of Leaf Spot Disease on Leafy Vegetable and Ornamental Crops Caused by Paramyrothecium and Albifimbria Species.

The genera Paramyrothecium and Albifimbria have been established from the former genus Myrothecium and they generally comprise common soil-inhabiting and saprophytic fungi. Within these genera, only two fungi have been recognized as phytopathogenic thus far: P. roridum and A. verrucaria, both of which cause necrotic leaf spots and plant collapse. Severe leaf necrosis and plant decay have been observed in Northern and Southern Italy on leafy vegetable crops. Thirty-six strains of Paramyrothecium- and Albifimbria-like fungi were isolated from affected plants belonging to eight different species. Based on morphological characteristics, 19 strains were assigned to A. verrucaria, whereas the remaining strains, which mostly resembled Paramyrothecium-like fungi, could not be identified precisely. Molecular characterization of six loci (internal transcribed spacer [ITS], ß-tubulin [tub2], calmodulin [cmdA], translation elongation factor 1-alpha [tef1], large subunit ribosomal RNA [LSU], and mitochondrial ATP 6synthase 6 [ATP6]) of the 36 new isolates and three previously ITS-characterized isolates assigned all strains to four species: A. verrucaria, P. roridum, P. foliicola, and P. nigrum. Single and concatenated phylogenetic analyses were conducted, and they clearly distinguished the isolated fungi into four different groups. A. verrucaria, P. roridum, P. foliicola, and P. nigrum were able to induce leaf necrosis singly, and they were confirmed to be the causal agents of the leaf spot disease through pathogenicity assays. The involvement of fungi previously considered saprophytic (i.e., P. foliicola and P. nigrum) in the development of plant disease for the first time deserves particular attention because of the possibility of their transmission by seeds and the limited knowledge of their management with chemicals.

Differentiation of Pythium spp. from vegetable crops with molecular markers and sensitivity to azoxystrobin and mefenoxam.

BACKGROUND: Pythium species attack various vegetable crops causing seed, stem and root rot, and 'damping-off' after germination. Pythium diseases are prevalently controlled by two classes of fungicides, QoIs with azoxystrobin and phenlyamides with mefenoxam as representatives. The present study aimed to test the sensitivity of six Pythium species from different vegetable crops to azoxystrobin and mefenoxam and differentiating species based on ITS, cytochrome b and RNA polymerase I gene sequences. RESULTS: The inter- and intra-species sensitivity to azoxystrobin was found to be stable, with the exception of one Pythium paroecandrum isolate, which showed reduced sensitivity and two cytochrome b amino acid changes. For mefenoxam, the inter-species sensitivity was quite variable and many resistant isolates were found in all six Pythium species, but no RNA polymerase I amino acid changes were observed in them. ITS and cytochrome b phylogenetic analyses permitted a clear separation of Pythium species corresponding to globose- and filamentous-sporangia clusters. CONCLUSION: The results document the necessity of well-defined chemical control strategies adapted to different Pythium species. Since the intrinsic activity of azoxystrobin among species was stable and no resistant isolates were found, it may be applied without species differentiation, provided it is used preventatively to also control highly aggressive isolates. For a reliable use of mefenoxam, precise identification and sensitivity tests of Pythium species are crucial because its intrinsic activity is variable and resistant isolates may exist. Appropriate mixtures and/or alternation of products may help to further delay resistance development. © 2018 Society of Chemical Industry.

Microbiological Parameters in the Primary Production of Berries: A Pilot Study.

The primary production of fresh soft fruits was considered to be a suspected critical point for the contamination of frozen berries that were responsible for the large 2013⁻2014 Hepatitis A virus (HAV) outbreak in Europe. In this study, an Italian berries’ production area was studied for its agro-technical characteristics, and the fresh fruits were analyzed for the presence of enteric viruses (HAV and Norovirus (NoV) genogroup I and genogroup II (GGI and GGII)), the enumeration of hygienic quality parameters, and the prevalence of bacterial pathogens. A total of 50 producers were sampled, who specialized in the exclusive or shared cultivation of berries. Escherichia coli was detected in two blackberry samples, whereas HAV and Norovirus were not detected. The samples were negative for Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC). The farms’ attributes were not associated with positive samples, apart from the presence of E. coli and the aerobic mesophilic bacteria for blackberry that were statistically correlated. In blueberries, the high aerobic mesophilic count could likely be associated with the resistance of the outer layer to handling. However, the two pathogens (Salmonella spp. and STEC) and the targeted viruses (HAV, NoV GGI and GGII) were not detected, highlighting the low risk of foodborne pathogens and viral contamination at the primary production stage of the berry food chain in the area considered in this pilot study.

Molecular phylogeny and characterization of secondary metabolite profile of plant pathogenic Alternaria species isolated from basil.

Alternaria leaf-spot is a new disease recently reported on basil in Italy. The correct identification of Alternaria species has suffered from many reclassifications in function of morphological features and molecular data. In our study, we performed an overall approach to obtain a better characterization of basil Alternaria isolates. Morphological characteristics, seven-genome region phylogenic analysis, and secondary metabolite profile differentiated the majority of the isolates as A. alternata. OPA 1-3 and OPA 10-2 were the best molecular regions to discriminate among the isolates. Morphological characteristics and sporulation groups helped to discriminate A. tenuissima from A. alternata isolates. All isolates in the A. sect. Alternaria were mycotoxigenic and pathogenic on basil, the production of mycotoxins was enhanced on basil compared to in vitro conditions used in this work.

Grapevine Grafting: Scion Transcript Profiling and Defense-Related Metabolites Induced by Rootstocks.

Rootstocks are among the main factors that influence grape development as well as fruit and wine composition. In this work, rootstock/scion interactions were studied using transcriptomic and metabolic approaches on leaves of the "Gaglioppo" variety, grafted onto 13 different rootstocks growing in the same vineyard. The whole leaf transcriptome of "Gaglioppo" grafted onto five selected rootstocks showed high variability in gene expression. In particular, significant modulation of transcripts linked to primary and secondary metabolism was observed. Interestingly, genes and metabolites involved in defense responses (e.g., stilbenes and defense genes) were strongly activated particularly in the GAG-41B combination, characterized in addition by the down-regulation of abscisic acid (ABA) metabolism. On the contrary, the leaves of "Gaglioppo" grafted onto 1103 Paulsen showed an opposite regulations of those transcripts and metabolites, together with the greater sensitivity to downy mildew in a preliminary in vitro assay. This study carried out an extensive transcriptomic analysis of rootstock effects on scion leaves, helping to unravel this complex interaction, and suggesting an interesting correlation among constitutive stilbenes, ABA compound, and disease susceptibility to a fungal pathogen.

Verrucarin A and roridin E produced on spinach by Myrothecium verrucaria under different temperatures and CO2 levels.

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.

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.

Production and release of asexual sporangia in Plasmopara viticola.

To study the influence of environmental conditions on sporulation of Plasmopara viticola lesions under vineyard's conditions, unsprayed vines were inspected every second or third day and the numbers of sporulating and nonsporulating lesions were counted in two North Italy vineyards in 2008 to 2010. Infected leaves were removed so that only fresh lesions were assessed at each field assessment. Sporulation was studied at two scales, across field assessments and across the seasonal population of lesions. Frequencies of sporulating lesions were positively correlated with the numbers of moist hours in the preceding dark period (i.e., the number of hours between 8:00 p.m. and 7:00 a.m. with relative humidity ≥80%, rainfall >0 mm, or wetness duration >30 min). In a receiver operating characteristic analysis, predicted sporulation based on the occurrence of ≥3 moist hours at night provided overall accuracy of 0.85. To study the time course of sporulation on lesions which were not washed by rainfall, numbers of sporangia produced per square millimeter of lesion were estimated on individual cohorts of lesions over the whole infectious period. The numbers of sporangia per square millimeter of lesion increased rapidly during the first 4 days after the beginning of sporulation and then tapered off prior to a halt; the time course of cumulative sporangia production by a lesion followed a monomolecular growth model (R(2) = 0.97). The total number of sporangia produced by a square millimeter of lesion increased as the maximum temperature decreased and moist hours in the dark increased. To study the release pattern of the sporangia, spore samplers were placed near grapevines with sporulating lesions. Airborne sporangia were caught in 91.2% of the days over a wide range of weather conditions, including rainless periods. The results of this study provide quantitative information on production of P. viticola sporangia that may help refine epidemiological models used as decision aids in grape disease management programs.

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.

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.