Endophytic Alternaria and Fusarium species associated to potato plants (Solanum tuberosum L.) in Iran and their capability to produce regulated and emerging mycotoxins.

Endophytic fungi live inside virtually every plant species, without causing any apparent disease or damage to the host. Nevertheless, under particular conditions, mutualistic lifestyle of endophytes may change to pathogenic. In this study, the biodiversity of Alternaria and Fusarium species, the two most abundant endophytic fungi isolated from healthy potato plants in two climatically different regions of Iran, Ardebil in the north-west and Kerman in the south-east, was investigated. Seventy-five Fusarium strains and 83 Alternaria strains were molecularly characterized by multi-locus gene sequencing. Alternaria strains were characterized by the sequences of gpd and caM gene fragments and the phylogenetic tree was resolved in 3 well-separated clades. Seventy-three strains were included in the clade A, referred as Alternaria section, 6 strains were included in clade B, referred as Ulocladioides section, and 4 strains were included in clade C, referred as Infectoriae section. Fusarium strains, identified by sequencing the translation elongation factor 1α (tef1), ß-tubulin (tub2) and internal transcribed spacer (ITS) genomic regions, were assigned to 13 species, viz. F. brachygibosum, F. clavum, F. equiseti, F. flocciferum, F. incarnatum, F. nirenbergiae, F. nygamai, F. oxysporum, F. proliferatum, F. redolens, F. sambucinum, F. solani and F. thapsinum. Twenty-six selected strains, representative of F. equiseti, F. nirenbergiae, F. oxysporum, F. nygamai, F. proliferatum, and F. sambucinum, were also tested for production of the mycotoxins deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin (T-2), beauvericin (BEA), enniatins (ENNs), fumonisins (FBs), fusaric acid (FA) and moniliformin (MON). None of the tested strains produced trichothecene toxins (DON, NIV, DAS and T-2). Two out of 2 F. equiseti isolates, 1/6 F. oxysporum, 1/3 F. proliferatum, and 1/9 F. nygamai did not produce any of the tested toxins; the rest of strains produced one or more BEA, ENNs, FBs, FA and MON toxins. The most toxigenic strain, F. nygamai ITEM-19012, produced the highest quantities of FBs (7946, 4693 and 4333 µg/g of B1, B2, and B3 respectively), along with the highest quantities of both BEA (4190 µg/g) and MON (538 µg/g). These findings suggest that contamination of potato tubers with mycotoxins in the field or at post-harvest, due to a change in lifestyle of endophytic microflora, should be carefully considered and furtherly investigated.

Phylogeny and mycotoxin profiles of pathogenic Alternaria and Curvularia species isolated from date palm in southern Tunisia.

Date palm (Phoenix dactylifera L.), is a widely cultivated crop across North Africa, with about 300 thousand tons of fruits produced per year, in Tunisia. A wide range of fungal pathogens has been associated with leaf spots of date palm, Alternaria species being the most frequently reported. Symptomatic leaves of Deglet Nour variety were randomly collected in six localities in Tunisia. We used a polyphasic approach to identify 45 Alternaria and five Curvularia strains isolated from date palm, confirming their pathogenicity. Sequencing of allergen Alt-a1, glyceraldehyde-3-phosphate dehydrogenase (gpd) and calmodulin genes allowed us to group 35 strains in Alternaria Section, and 10 strains in Ulocladioides section. Based on sequencing analyses of Internal Transcribed Spacer, gpd and elongation factor genomic regions, all Curvularia strains were identified as Curvularia spicifera. All Alternaria and Curvularia species tested on date palm plantlets proved to be pathogenic, fulfilling Koch's postulates. Although no significant differences were observed among the species, the highest mean disease severity index was observed in A. arborescens, while the lowest corresponded to C. spicifera. The capability of these strains to produce mycotoxins in vitro was evaluated. None of the A. consortialis strains produced any known Alternaria mycotoxin, whereas more than 80% of the strains included in Alternaria section Alternaria produced variable amounts of multiple mycotoxins such as alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid and tentoxin. Curvularia spicifera strains produced detectable traces of fumonisins B. This work reports a first comprehensive multidisciplinary study of mycotoxigenic Alternaria species and C. spicifera associated with leaf spot disease on date palm.

Deoxynivalenol and T-2 Toxin as Major Concerns in Durum Wheat from Italy.

Fusarium Head Blight is a devastating disease of wheat caused by a complex of Fusarium species producing a wide range of mycotoxins. Fusarium species occurrence is variable in different geographical areas and subjected to a continuous evolution in their distribution. A total of 141 durum wheat field samples were collected in different regions of Italy in three years, and analyzed for Fusarium species and related mycotoxin occurrence. Mycotoxin contamination varied according to year and geographical origin. The highest mycotoxin contamination was detected in 2014. Deoxynivalenol was detected with an average of 240 µg/kg only in Central and Northern Italy; and T-2 and HT-2 toxins with an average of 150 µg/kg in Southern Italy. Approximately 80% of samples from Southern Italy in 2013/2014 showed T-2 and HT-2 levels over the EU recommended limits. Fusarium graminearum occurred mostly in Northern Italy, while F. langsethiae occurred in Southern Italy. These data showed that a real mycotoxin risk related to Fusarium exists on the whole in Italy, but varies according with geographical areas and environmental conditions. Consistent monitoring of Fusarium species and related mycotoxin distribution on a long period is worthwhile to generate more accurate knowledge on Fusarium species profile and mycotoxins associated and better establish the climatic change impact on wheat Fusarium epidemiology.

Diversity of Fusarium Species Isolated from Symptomatic Plants Belonging to a Wide Range of Agri-Food and Ornamental Crops in Lebanon.

Lebanon is a small Mediterranean country with different pedoclimatic conditions that allow the growth of both temperate and tropical plants. Currently, few studies are available on the occurrence and diversity of Fusarium species on Lebanese crops. A wide population of Fusarium strains was isolated from different symptomatic plants in the last 10 years. In the present investigation, a set of 134 representative strains were molecularly identified by sequencing the translation elongation factor, used in Fusarium as a barcoding gene. Great variability was observed, since the strains were grouped into nine different Fusarium Species Complexes (SCs). Fusarium oxysporum SC and Fusarium solani SC were the most frequent (53% and 24%, respectively). Members of important mycotoxigenic SCs were also detected: F. fujikuroi SC (7%), F. sambucinum SC (5%), F. incarnatum-equiseti SC (3%), and F. tricinctum SC (4%). Two strains belonging to F. lateritium SC, a single strain belonging to F. burgessii SC, and a single strain belonging to F. redolens SC were also detected. This paper reports, for the first time, the occurrence of several Fusarium species on Lebanese host plants. The clear picture of the Fusarium species distribution provided in this study can pose a basis for both a better understanding of the potential phytopathological and toxicological risks and planning future Fusarium management strategies in Lebanon.

Occurrence and Characterization of Penicillium Species Isolated from Post-Harvest Apples in Lebanon.

The apple is one of the most important fruit tree crops in the Mediterranean region. Lebanon, in particular, is among the top apple producer countries in the Middle East; however, recently, several types of damage, particularly rot symptoms, have been detected on fruits in cold storage. This study aims to identify the causal agents of apple decay in Lebanese post-harvest facilities and characterize a set of 39 representative strains of the toxigenic fungus Penicillium. The results demonstrated that blue mould was the most frequent fungal disease associated with apples showing symptoms of decay after 3-4 months of storage at 0 °C, with an average frequency of 76.5% and 80.6% on cv. Red and cv. Golden Delicious apples, respectively. The morphological identification and phylogenetic analysis of benA gene showed that most Penicillium strains (87.2%) belong to P. expansum species whereas the remaining strains (12.8%) belong to P. solitum. Furthermore, 67.7% of P. expansum strains produced patulin when grown on apple puree for 14 days at 25 °C with values ranging from 10.7 mg kg-1 to 125.9 mg kg-1, whereas all P. solitum did not produce the mycotoxin. This study highlights the presence of Penicillium spp. and their related mycotoxin risk during apple storage and calls for the implementation of proper measures to decrease the risk of mycotoxin contamination of apple fruit products.

Plasma Technology Increases the Efficacy of Prothioconazole against Fusarium graminearum and Fusarium proliferatum Contamination of Maize (Zea mays) Seedlings.

The contamination of maize by Fusarium species able to produce mycotoxins raises great concern worldwide since they can accumulate these toxic metabolites in field crop products. Furthermore, little information exists today on the ability of Fusarium proliferatum and Fusarium graminearum, two well know mycotoxigenic species, to translocate from the seeds to the plants up to the kernels. Marketing seeds coated with fungicide molecules is a common practice; however, since there is a growing need for reducing chemicals in agriculture, new eco-friendly strategies are increasingly tested. Technologies based on ionized gases, known as plasmas, have been used for decades, with newer material surfaces, products, and approaches developed continuously. In this research, we tested a plasma-generated bilayer coating for encapsulating prothioconazole at the surface of maize seeds, to protect them from F. graminearum and F. proliferatum infection. A minimum amount of chemical was used, in direct contact with the seeds, with no dispersion in the soil. The ability of F. graminearum and F. proliferatum species to translocate from seeds to seedlings of maize has been clearly proven in our in vitro experiments. As for the use of plasma technology, the combined use of the plasma-generated coating with embedded prothioconazole was the most efficient approach, with a higher reduction of the infection of the maize seminal root system and stems. The debated capability of the two Fusarium species to translocate from seeds to seedlings has been demonstrated. The plasma-generated coating with embedded prothioconazole resulted in a promising sustainable approach for the protection of maize seedlings.

Mycotoxin Profile and Phylogeny of Pathogenic Alternaria Species Isolated from Symptomatic Tomato Plants in Lebanon.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani, A. alternata, and A. arborescens. A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A.limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg-1, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.

Phylogeny and mycotoxin profile of Fusarium species isolated from sugarcane in Southern Iran.

Sugarcane is an important crop in Southern Iran for agri-food, energy, and pharmaceutical industries. Among the pathogens that colonize sugarcane, mycotoxigenic Fusarium species are reason of serious concern for both their pathogenicity on plants and ability to produce harmful mycotoxins to humans and animals. We studied 104 Fusarium strains, selected within a wider Fusarium set isolated from sugarcane in Southern Iran, for molecular identification, phylogeny and mycotoxin analyses. Most of Fusarium strains belonged to Fusarium fujikuroi Species Complex (FFSC) and identified mainly as F. proliferatum, at minor extent as F. sacchari, and rarely as F. thapsinum, and F. verticillioides. Moreover, 14 strains identified as FFSC could not be assigned to any known species, although they were phylogenetically closely related to F. andiyazi, likely representing a new phylogenetic species. A subset of FFSC strains were analyzed for in vitro production of fumonisins (FBs), beauvericin (BEA), and enniatins (ENNs). Fusarium proliferatum strains produced FBs at high amount, and, at a lesser extent, BEA, and ENNs; F.sacchari produced only BEA and B ENNs at very low level; Fusarium sp. strains produced only B ENNs. The paper provides new insights on the genetic diversity of Fusarium species and their mycotoxin profile occurring on sugarcane in Iran.

Phylogeny and Mycotoxin Profile of Pathogenic Fusarium Species Isolated from Sudden Decline Syndrome and Leaf Wilt Symptoms on Date Palms (Phoenix dactylifera) in Tunisia.

In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, Fusarium was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative Fusarium strains at species level and investigate their phylogenetic relationships. The main species detected was Fusariumproliferatum, and at a much lesser extent, Fusariumbrachygibbosum, Fusariumcaatingaense, Fusariumclavum, Fusariumincarnatum, and Fusariumsolani. Pathogenicity on the DegletNour variety plantlets and the capability to produce mycotoxins were also assessed. All Fusarium species were pathogenic complying Koch's postulates. Fusariumproliferatum strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All F.brachygibbosum strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. Fusariumcaatingaense, F.clavum, F.incarnatum produced only BEA. Fusariumsolani strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of Fusarium species on date palms, concerning both phytopathological and food safety issues.

Genetic structure of Fusarium verticillioides populations from maize in Iran.

Fusarium verticillioides is one of the most important fungal pathogens of maize since it causes severe yield losses and produces the mycotoxins fumonisins that represent a major concern for human and animal health. Information about genetic diversity and population structure of fungal pathogens is essential for developing disease management strategies. The aim of this research was to investigate the genetic structure of F. verticillioides isolated from different provinces of Iran through determination of mating type idiomorphs, phylogenetic analyses based on translation elongation factor-1 alpha (EF-1α), RNA Polymerase II Subunit (RPB2), beta-tubulin (tub2) and Calmodulin (cmdA) genes and genetic diversity analyses based on 6 simple-sequence repeats (SSRs). Both mating types were detected in Iranian populations of F. verticillioides, particularly in Qazvin and Khuzestan, with equal frequency, which highlighted that sexual reproduction is favorable under field conditions. However, the linkage disequilibrium indices did not support the hypothesis of random mating in Khuzestan and Fars. Although assessment of nucleotide diversity based on housekeeping genes showed low level of variation among strains, genotype diversity based on SSRs revealed a high level of genetic diversity within Iranian populations. AMOVA analysis highlighted that the genetic variation of F. verticillioides in Iran was mainly distributed within population of a single area (97%), while a small proportion of genetic variation (3%) resided among populations. These patterns of variation are likely explained by the continuous gene flow among populations isolated from different areas. On the other hand, principal coordinate analysis indicated that the distribution of genetic variation among populations could be explained by the geographical distances. Consequently, to reduce pathogen gene flow among regions, the quarantine processes in Iran should be intensified.

First report of Leaf Wilt caused by Fusarium proliferatum and F. brachygibbosum on Date Palm (Phoenix dactylifera) in Tunisia.

Since 2017, a new leaf wilt syndrome was observed in plantations of date palm in Tunisia. Its incidence increases sharply from year to year, especially in 'Deglet Nour' trees, aged between 5 and 15 years. In severe cases, the large number of dried leaves per tree can lead to complete cessation of date production. Symptoms appear on one or more leaves in the center of the crown. Whitening and drying start at the top of the leaflets and proceed to their base, while the midrib remains green. Then the whole leaf dies. Small white-creamy leaflet fragments and roots were collected from five different regions in the Djerid Oases. They were disinfected with diluted bleach (0,8 % NaOCl) and ethanol (80%) (each 2 min), rinsed with sterile distilled water, dried and finally plated in Petri dishes containing Potato Dextrose Agar (PDA) amended with 50mg/l neomycin. After incubation for 7 days at 25ºC±2, emerging fungal colonies were single-spored by serial dilution. They were transferred to PDA, Carnation Leaf Agar (CLA) and Spezieller Nahrstoffarmer Agar (SNA) for morphological identification. Based on the colony color on PDA, conidial morphology and phialide structures on CLA and/or SNA, of the 85 Fusarium isolates, around 90% were identified as F. proliferatum and around 10% as F. brachygibbosum (Leslie and Summerell, 2006). Fusarium proliferatum colonies rapidly developed white aerial mycelium that became purple in old cultures. Microconidia were abundant in the aerial mycelium and formed chains of variable length, on monophialides and polyphialids, a characteristic that distinguishes F. proliferatum from F. verticilloides. Less often, they were observed in false heads. Chlamydospores were absent. On CLA, microconidia were mostly 2 × 15 µm in size, a large number of sickle shaped macroconidia (2 × 25 µm) had one septum, some were larger (2 × 50 µm) with 3 septa and tips at both ends. Molecular identification was carried out based on elongation factor (EF-1α) gene sequencing. The region between the EF1 and EF2 primers (O'Donnell et al., 1998) was amplified and the sequences were compared to Fusarium reference sequences (GenBank). The sequences of the isolates Fus 1953 (539 bp), Fus 1962 (618 bp), and Fus 1965 (605 bp) shared respectively 100%, 99.51% and 99.51% homology with that of F. proliferatum JF740713.1 and were deposited in GenBank with the following accession numbers: MT630418, MT630419, and MT630420, respectively. The sequences of isolates 7F, 28F, Fus 1955 and Fus 1956 shared 100 % homology with that of F. brachygibbosum (GQ505418.1) while those of Fus 1955 and Fus 1956 showed 99.02 and 98.91 % identity, respectively, with F. brachygibbosum JX118981.1. The sequences of 7F (535 bp), 28F (535 bp), Fus 1955 (608 bp), and Fus 1956 (647 bp) were deposited in GenBank with the following accession numbers: MT630409, MT630410, MT630411, and MT630412, respectively. Two ml suspension of 106 conidia / ml of each isolate was sprayed separately or in combinations on in vitro cloned 'Deglet Nour' plants, placed in a greenhouse at 28°±2 °C and 70% R.H.. Isolates of F. proliferatum led to dryness and wilting leaflets after 3 weeks. Fusarium brachygibbosum only induced mild leaf yellowing, while in combination they were more virulent. Fungal isolates of both species were re-isolated and their identity confirmed to be the same of those isolated from leaflets infected in the open field, confirming Koch's postulates. Control plants lacked symptoms. Fusarium proliferatum is known as date palm pathogen in many countries (Saleh et al. 2017), however, to our knowledge, this is the first report of F. proliferatum and also F. brachygibbosum causing Leaf Wilt symptoms on P. dactylifera in Tunisia.

Molecular Identification and Mycotoxin Production by Alternaria Species Occurring on Durum Wheat, Showing Black Point Symptoms.

Black point is a fungal disease of wheat, mainly associated with mycotoxigenic Alternaria species. Affected wheat kernels are characterized by dark brown discolouration of the embryo region and reduction of grain quality. Potential risk is the possible accumulation of Alternaria mycotoxins, alternariol (AOH), alternariol-monomethyl ether (AME), tenuazonic acid (TA), and altenuene (ALT), provided by haemato-toxic, genotoxic, and mutagenic activities. One hundred and twenty durum wheat samples belonging to 30 different genotypes grown in Bologna and Modena areas, in Italy, showing black point symptoms, were analyzed for Alternaria species and their mycotoxin contamination. Alternariol was selected as an indicator of the capability of the Alternaria species to produce mycotoxin in vivo in field conditions. The data showed that Alternaria species occurred in 118 out of 120 wheat kernels samples, with the incidence of infected kernels ranging between 1% and 26%. Moreover, AOH was detected by using a HPLC with a diode array detector (LC-DAD) in 98 out of 120 samples with values ranging between 24 and 262 µg Kg-1. Ninety-two Alternaria representative strains, previously identified morphologically, were identified at species/section level using gene sequencing, and therefore were analyzed for their mycotoxin profiles. Eighty-four strains, phylogenetically grouped in the Alternaria section, produced AOH, AME, and TA with values up to 8064, 14,341, and 3683 µg g-1, respectively, analyzed by using a LC-DAD. On the other hand, eight Alternaria strains, included in Infectoriae Section, showed a very low or no capability to produce mycotoxins.

Antagonistic activity of olive endophytic bacteria and of Bacillus spp. strains against Xylella fastidiosa.

Strains of Xylella fastidiosa subsp. pauca characterized by a specific genotype, the so called sequence type "ST53", have been associated with a severe disease named Olive Quick Decline Syndrome (OQDS). Despite the relevant research efforts devoted to control the disease caused by X. fastidiosa, so far there are no therapeutic means able to cure the infected host plants. As such, the aim of this study was the identification of antagonistic bacteria potentially deployable as bio-control agents against X. fastidiosa. To this end, two approaches were used, i.e. the evaluation of the antagonistic activity of: i) endophytic bacteria isolated from olive trees located in an infected area but showing mild or no symptoms, and ii) Bacillus strains, as they are already known as bio-control agents. Characterization of endophytic bacterial isolates revealed that the majority belonged to different species of the genera Sphingomonas, Methylobacterium, Micrococcus and Curtobacterium. However, when they were tested in vitro against X. fastidiosa ST53 none of them showed antagonistic activity. On the contrary, when strains belonging to different species of the genus Bacillus were included in these tests, remarkable antagonistic activities were recorded. Some B. velezensis strains also produced culture filtrates with inhibitory activity against X. fastidiosa ST53. Taking also into account that two of these B. velezensis strains (namely strains D747 and QST713) are already registered and commercially available as bio-control agents, our results pave the way for further studies aimed at the development of a sustainable bio-control strategy of the OQDS.

Alternaria species associated to wheat black point identified through a multilocus sequence approach.

Black point is one of the most important wheat disease and its incidence is increasing worldwide due to climate change too. Among the fungal genera that can cause black point, Alternaria is one of the predominant genus, often associated with mycotoxin contamination. The correct identification is the baseline for prevention and control of the disease. Taxonomy of the genus Alternaria is not completely clear yet, since its species can be differentiated for few morphological traits and, in some cases, also molecular phylogeny is not very effective in establishing species boundaries. In this study, one-hundred sixty-four strains, isolated from wheat kernels affected by black point sampled worldwide, were analyzed in order to assess their identity. Sequences of elongation factor, ß-tubulin, glyceraldehyde-3-phosphate dehydrogenase and allergen alt-a1 genes were used to identify the variability of this population and their phylogenetic relationships. Isolates were grouped in two main clades: the Alternaria section, including A. alternata, A. tenuissima and A. arborescens species, and the Infectoriae section, that includes the two species A. infectoria and A. triticina. Comparison of isolates according with their area of isolation did not show a correlation between phylogeny and geographic origin. Indeed, the isolates grouped on the base of only their phylogenetic relationship. Due to the data arisen by our study, we strongly recommend a multilocus sequence approach to define Alternaria species, based on common genes and procedures to be unanimously shared by scientific community dealing with Alternaria genus. Moreover, we suggest that A. alternata, A. tenuissima, A. turkisafria and A. limoniasperae species would be merged in the defined species A. alternata. Finally we recommend to consider a taxonomic re-evaluation of the Infectoriae section that, for the morphology, sexuality, genetic and mycotoxin profile of the species included, could be defined as different fungal genus from Alternaria.

In Vitro and in Field Response of Different Fungicides against Aspergillus flavus and Fusarium Species Causing Ear Rot Disease of Maize.

Aspergillus flavus, the main aflatoxin B1 producing fungal species, Fusarium graminearum, a deoxynivalenol producer, and the fumonisin-producing species F. proliferatum and F. verticillioides are the main toxigenic fungi (TF) that colonize maize. Several strategies are available to control TF and related mycotoxins, such as chemical control. However, there is poor knowledge on the efficacy of fungicides on maize plants since few molecules are registered. The sensitivity of F. graminearum, F. proliferatum, F. verticillioides, and A. flavus to eleven fungicides, selected based on their different modes of action, was evaluated in both in vitro assays and, after selection, in the field. In vitro, demethylation inhibitors (DMI) showed excellent performances, followed by thiophanate-methyl and folpet. Among the succinate dehydrogenase inhibitors (SDHI), isopyrazam showed a higher effectiveness against Fusarium species than boscalid, which was ineffective against Fusarium, like the phenyl-pyrrole fludioxonil. Furthermore, both SDHIs and fludioxonil were more active against A. flavus than Fusarium species. In field trials, prothioconazole and thiophanate-methyl were confirmed to be effective to reduce F. graminearum (52% and 48%) and F. proliferatum contamination (44% and 27%). On the other hand, prothioconazole and boscalid could reduce A. flavus contamination at values of 75% and 56%, respectively.

Phylogeny and Mycotoxin Characterization of Alternaria Species Isolated from Wheat Grown in Tuscany, Italy.

Wheat, the main source of carbohydrates worldwide, can be attacked by a wide number of phytopathogenic fungi, included Alternaria species. Alternaria species commonly occur on wheat worldwide and produce several mycotoxins such as tenuazonic acid (TA), alternariol (AOH), alternariol-monomethyl ether (AME), and altenuene (ALT), provided of haemato-toxic, genotoxic, and mutagenic activities. The contamination by Alternaria species of wheat kernels, collected in Tuscany, Italy, from 2013 to 2016, was evaluated. Alternaria contamination was detected in 93 out of 100 field samples, with values ranging between 1 and 73% (mean of 18%). Selected strains were genetically characterized by multi-locus gene sequencing approach through combined sequences of allergen alt1a, glyceraldeyde-3-phosphate dehydrogenase, and translation elongation factor 1α genes. Two well defined groups were generated; namely sections Alternaria and Infectoriae. Representative strains were analyzed for mycotoxin production. A different mycotoxin profile between the sections was shown. Of the 54 strains analyzed for mycotoxins, all strains included in Section Alternaria produced AOH and AME, 40 strains (99%) produced TA, and 26 strains (63%) produced ALT. On the other hand, only a very low capability to produce both AOH and AME was recorded among the Section Infectoriae strains. These data show that a potential mycotoxin risk related to the consumption of Alternaria contaminated wheat is high.

Molecular characterisation and detection of resistance to succinate dehydrogenase inhibitor fungicides in Botryotinia fuckeliana (Botrytis cinerea).

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs), interfering with fungal respiration, are considered to be fungicides at medium to high risk of resistance. Boscalid was the first molecule belonging to the SDHIs that was introduced for the control of Botryotinia fuckeliana. A range of different target-site mutations leading to boscalid resistance have been found in field populations of the fungus. The different types of mutation confer different cross-resistance profiles towards novel SDHIs, such as the recently introduced fungicide fluopyram. This study combines the determination of cross-resistance profiles and the setting-up of methods for fast molecular detection of the mutations. RESULTS: By means of in vitro tests, a range of SdhB mutations were characterised for resistance levels towards boscalid and fluopyram. SdhB mutations conferring P225L and P225F substitutions conferred high resistance to boscalid and high or moderate resistance to fluopyram respectively. Mutants carrying the N230I replacement were moderately resistant to both SDHIs. Substitutions at position H272 responsible for a high level of resistance to boscalid conferred sensitivity (H272R), hypersensitivity (H272Y) or moderate resistance (H272V) to fluopyram. Allele-specific (AS) PCR was developed and used for genotyping 135 B. fuckeliana isolates. The assay confirmed the strict association between resistance profiles and allelic variants of the SdhB gene. Real-time AS-PCR proved to be sensitive and specific for quantitative detection of different SDHI-resistant genotypes. CONCLUSION: Fluopyram-resistant mutants are currently rarely detected in the field sprayed with boscalid, but this may change with intensive exposure of the fungal population to fluopyram. PCR assays/methods developed in the study provide tools for fast monitoring of field populations and observing possible changes in population composition following fluopyram introduction, useful for the setting-up of appropriate preventive measures.

Occurrence of fungicide resistance in populations of Botryotinia fuckeliana (Botrytis cinerea) on table grape and strawberry in southern Italy.

BACKGROUND: Botryotinia fuckeliana (Botrytis cinerea) is a pathogen with a high risk of development of resistance to fungicides. Fungicide resistance was monitored during 2008-2011 in B. fuckeliana populations from both table-grape vineyards and greenhouse-grown strawberries in southern Italy. RESULTS: Isolates showing different levels of resistance to anilinopyrimidines (APs) were detected at high frequency (up to 98%) in fields treated intensively with APs (4-7 sprays season(-1) ). A slight decrease in sensitivity to fludioxonil, always combined with AP resistance, was generally found at lower frequencies. The repeated use of fenhexamid on grapevine (3-8 sprays season(-1) ) led to a strong selection of highly resistant isolates (up to 100%). Boscalid-resistant mutants were detected at very variable frequencies (0-73%). Occurrence of resistance to quinone outside inhibitors (QoIs) was also ascertained. Multiple fungicide resistance to 2-6 different modes of action were frequently recovered. Single nucleotide polymorphisms (SNPs) in the target genes Erg27, SdhB and cytb were associated with resistance to fenehexamid, boscalid and QoIs respectively. CONCLUSION: Resistance to the fungicides commonly used against grey mould on table grape and strawberry is quite common in southern Italy. This is an outcome of the incorrect use of fungicides, often because of the maximum number of detectable residues of plant protection products imposed by big international retailers, and underlines the crucial role of antiresistance strategies in integrated pest management.

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides.

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild-type strains, or from naturally infected plants on a medium amended with 1-3 mg L(-1) trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved.