Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower.

BACKGROUND: The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population. RESULTS: In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus. CONCLUSION: The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries.

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and ß-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Pythium banihashemianum sp. nov. and Globisporangium izadpanahii sp. nov.: Two New Oomycete Species from Rice Paddies in Iran.

An investigation into oomycete diversity in rice paddies of Fars Province in Iran led to the identification of two new Pythium sensu lato (s.l.) species as Globisporangium izadpanahii sp. nov. and Pythium banihashemianum sp. nov. The identification was based on morphological and physiological features as well as on the phylogenetic analysis of nuclear (ITS and ßtub) and mitochondrial (cox1 and cox2) loci using Bayesian inference and Maximum Likelihood. The present paper formally describes these two new species and defines their phylogenetic relationships with other congeneric species. According to multiple gene genealogy analysis, G. izadpanahii sp. nov. was grouped with other species of Globisporangium (formerly, clade G of Pythium s.l.) and was closely related to both G. nagaii and the recently described G. coniferarum. The second species, designated P. banihashemianum sp. nov., was grouped with other species of Pythium sensu stricto (formerly, clade B of Pythium s.l.) and, according to the phylogenetic analysis, shared an ancestor with P. plurisporium. The production of globose hyphal swellings was a major characteristic of G. izadpanahii sp. nov., which did not produce vesicles and zoospores. In pathogenicity tests on rice seedlings, P. banihashemianum sp. nov. isolates were highly pathogenic and caused severe root and crown rot, while G. izadpanahii sp. nov. isolates were not pathogenic.

Trunk Injection Delivery of Biocontrol Strains of Trichoderma spp. Effectively Suppresses Nut Rot by Gnomoniopsis castaneae in Chestnut (Castanea sativa Mill.).

Gnomoniopsis castaneae is responsible for brown or chalky nut rot in sweet chestnut (Castanea sativa), causing heavy reductions in nut production. Controlling it is challenging, due to its inconspicuous infections, erratic colonization of host tissues and endophytic lifestyle. Fungicides are not applicable because they are prohibited in chestnut forests and strongly discouraged in fruit chestnut groves. Trichoderma species are safe and wide-spectrum biocontrol agents (BCAs), with a variety of beneficial effects in plant protection. This study tested selected strains of T. viride, T. harzianum and T. atroviride for their ability to suppress G. castaneae. Field experiments were conducted in four chestnut groves (two test plots plus two controls) at two sites with a different microclimate. As the size of the trees were a major drawback for uniform and effective treatments, the Trichoderma strains were delivered directly by trunk injection, using the BITE® (Blade for Infusion in TrEes) endotherapic tool. The BCA application, repeated twice in two subsequent years, significantly reduced nut rot incidence, with a more marked, presumably cumulative, effect in the second year. Our data showed the tested Trichoderma strains retain great potential for the biological control of G. castaneae in chestnut groves. The exploitation of Trichoderma spp. as biopesticides is a novelty in the forestry sector and proves the benefits of these microbes in plant disease protection.

Chitosan-based films grafted with citrus waste-derived antifungal agents: An innovative and sustainable approach to enhance post-harvest preservation of citrus fruit.

This paper reports the synthesis, characterization, and properties of chitosan films (CHI) grafted with a natural antifungal agent with the aim of developing active films of natural origin to prevent post-harvest losses of citrus fruit. The antifungal agent was prepared by fermentation using lemon peel (AntiFun-LM), a citrus waste, and grafted on chitosan using different coupling agents (CHI/AntiFun-LM). Bioactive films were prepared by solvent casting. FTIR-ATR and ToF-SIMS analyses provided compelling evidence of the successful grafting process. TGA-DSC demonstrated that the films are stable after grafting. SEM studies showed the continuous and compact surface of the films. WCA measurements proved that CHI/AntiFun-LM films are more hydrophilic than CHI films. Moreover, the CHI/AntiFun-LM films showed stronger UV shielding effect when compared to CHI. The biological evaluation demonstrated that CHI/AntiFun-LM films gained considerable antifungal properties against most fungi responsible for post-harvest decay. Cytotoxicity tests showed that CHI/AntiFun-LM films did not cause any toxic effect against L929 fibroblasts. This study highlights the great potential of chemical grafting of antifungal agents produced from citrus waste to chitosan and preparation of natural-based films to act as a powerful alternative in post-harvest protection of citrus fruit in a perspective of circular economy.

Phytophthora × pelgrandis Causes Root and Collar Rot of Lavandula stoechas in Italy.

In 2007, Phytophthora isolates with atypical morphological and biological characteristics were found associated with root and collar rot of potted plants of Stoechas lavender (Lavandula stoechas) in an ornamental nursery in Italy. A polyphasic approach, including morphological and cultural observations, sequencing the ITS-rDNA region, the Pheca and the mitochondrial coxI genes, multiplex PCRs with primers specific for P. nicotianae or P. cactorum, as well as random amplified polymorphic DNA-polymerase chain reaction, was used to characterize these isolates. On the basis of morpho-cultural and molecular analyses, the isolates from Stoechas lavender were identified as Phytophthora × pelgrandis, a natural hybrid of P. nicotianae × P. cactorum previously reported in other European countries, the Americas, and Taiwan, as a pathogen of ornamentals and loquat plants. In pathogenicity tests using potted plants of Stoechas lavender, the P. × pelgrandis isolates, similarly to the parental species P. nicotianae, induced the symptoms observed on plants with natural infections and were reisolated only from artificially inoculated plants. Dispersal of P. × pelgrandis on this host could exacerbate the damage caused by Phytophthora root and collar rot, of which the main causal agent presently is P. nicotianae on lavender in Europe. Application of hygienic measures is important to reduce the proliferation and spread of the Phytophthora hybrids.

Secondary metabolites produced by four Colletotrichum species in vitro and on fruits of diverse olive cultivars.

This study was aimed to characterize the secondary metabolites produced by four Colletotrichum species, C. acutatum, C. gloeosporioides, C. godetiae and C. karsti, both in vitro, on potato dextrose agar (PDA) and oatmeal agar (OA), and during the infection process of fruits of four olive cultivars differing in susceptibility to anthracnose, 'Coratina' and 'Ottobratica', both susceptible, 'Frantoio' and 'Leccino', both resistant. The metabolites were extracted from axenic cultures after seven days incubation and from olives inoculated singularly with each Colletotrichum species, at three different times, 1, 3 and 7 days post inoculation (dpi). They were identified using the UHPLC-QTOF-MS analysis method. In total, as many as 45 diverse metabolites were identified. Only 10 metabolites were present in both fruits and axenic cultures while 19 were found exclusively on olives and 16 exclusively in axenic cultures. The identified metabolites comprised fatty acid, phenolics, pyrones, sterols, terpenes and miscellaneous compounds. Each Colletotrichum species produced a different spectrum of metabolites depending on the type of matrices. On artificially inoculated olives the severity of symptoms, the amount of fungal secondary metabolites and their number peaked 7 dpi irrespective of the cultivar susceptibility and the virulence of the Colletotrichum species.

Molecular Variability of the Fusarium solani Species Complex Associated with Fusarium Wilt of Melon in Iran.

Species of the Fusarium solani species complex (FSSC) are responsible for the Fusarium wilt disease of melon (Cucumis melo), a major disease of this crop in Iran. According to a recent taxonomic revision of Fusarium based primarily on multilocus phylogenetic analysis, Neocosmospora, a genus distinct from Fusarium sensu stricto, has been proposed to accommodate the FSSC. This study characterized 25 representative FSSC isolates from melon collected in 2009-2011 during a field survey carried out in five provinces of Iran. Pathogenicity assays showed the isolates were pathogenic on different varieties of melon and other cucurbits, including cucumber, watermelon, zucchini, pumpkin, and bottle gourd. Based on morphological characteristics and phylogenetic analysis of three genetic regions, including nrDNA internal transcribed spacer (ITS), 28S nrDNA large subunit (LSU) and translation elongation factor 1-alpha (tef1), Neocosmospora falciformis (syn. F. falciforme), N. keratoplastica (syn. F. keratoplasticum), N. pisi (syn. F. vanettenii), and Neocosmospora sp. were identified among the Iranian FSSC isolates. The N. falciformis isolates were the most numerous. This is the first report of N. pisi causing wilt and root rot disease in melon. Iranian FSSC isolates from different regions in the country shared the same multilocus haplotypes suggesting a long-distance dispersal of FSSC, probably through seeds.

Genetic Variation of Puccinia triticina Populations in Iran from 2010 to 2017 as Revealed by SSR and ISSR Markers.

Puccinia triticina is a major wheat pathogen worldwide. Although Iran is within the Fertile Crescent, which is supposed to be the center of origin of both wheat and P. triticina, the knowledge of the genetic variability of local populations of this basidiomycete is limited. We analyzed 12 inter simple sequence repeats (ISSRs) and 18 simple sequence repeats (SSRs) of 175 P. triticina isolates sampled between 2010 and 2017 from wheat and other Poaceae in 14 provinces of Iran. SSRs revealed more polymorphisms than ISSRs, indicating they were more effective in differentiating P. triticina populations. Based on a dissimilarity matrix with a variable mutation rate for SSRs and a Dice coefficient for ISSRs, the isolates were separated into three large groups, each including isolates from diverse geographic origins and hosts. The grouping of SSR genotypes in UPGMA dendrograms was consistent with the grouping inferred from the Bayesian approach. However, isolates with a common origin clustered into separate subgroups within each group. The high proportion of heterozygous alleles suggests that in Iran clonal reproduction prevails over sexual reproduction of the pathogen. A significant correlation was found between SSR and ISSR genotypes and the virulence phenotypes of the isolates, as determined in a previous study.

Defense Mechanisms Induced by Celery Seed Essential Oil against Powdery Mildew Incited by Podosphaera fusca in Cucumber.

This study aimed to evaluate the effectiveness of essential oil extracted from celery (Apium graveolens) seeds (CSEO) for the control of powdery mildew of cucumber (Cucumis sativus) incited by Podosphaera fusca and to investigate the metabolic and genetic defense mechanisms triggered by the treatment with this essential oil in cucumber seedlings. The main compounds in the CSEO as determined by gas chromatography-mass spectrometry (GC-MS) analysis were d-limonene, 3-butyl phthalide, ß-selinene, and mandelic acid. The treatment with CSEO led to an increase in the content of both chlorophyll and phenolic/flavonoid compounds in cucumber leaves. In greenhouse tests, the application of CSEO reduced by 60% the disease severity on leaves of cucumber plants and stimulated the activity of defense-related enzymes such as ß-1,3-glucanase, chitinase, phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase. Moreover, treatment with CSEO induced overexpression of ß-1,3-glucanase, chitinase, and phenylalanine ammonia-lyase genes. A highly significant correlation was found between the ß-1,3-glucanase, chitinase, and phenylalanine ammonia-lyase enzymatic activities and the relative expression of the corresponding encoding genes in both inoculated and non-inoculated cucumber seedlings treated with the essential oil. Overall, this study showed that CSEO is a promising eco-friendly candidate fungicide that can be exploited to control cucumber powdery mildew.

Diversity of Mycotoxins and Other Secondary Metabolites Recovered from Blood Oranges Infected by Colletotrichum, Alternaria, and Penicillium Species.

This study identified secondary metabolites produced by Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium digitatum in fruits of two blood orange cultivars before harvest. Analysis was performed by UHPLC-Q-TOF-MS. Three types of fruits were selected, asymptomatic, symptomatic showing necrotic lesions caused by hail, and mummified. Extracts from peel and juice were analyzed separately. Penicillium digitatum was the prevalent species recovered from mummified and hail-injured fruits. Among 47 secondary metabolites identified, 16, 18, and 13 were of A. alternata, C. gloeosporioides, and P. digitatum, respectively. Consistently with isolations, indicating the presence of these fungi also in asymptomatic fruits, the metabolic profiles of the peel of hail-injured and asymptomatic fruits did not differ substantially. Major differences were found in the profiles of juice from hail-injured and mummified fruits, such as a significant higher presence of 5,4-dihydroxy-3,7,8-trimethoxy-6C-methylflavone and Atrovenetin, particularly in the juice of mummified fruits of the Tarocco Lempso cultivar. Moreover, the mycotoxins patulin and Rubratoxin B were detected exclusively in mummified fruits. Patulin was detected in both the juice and peel, with a higher relative abundance in the juice, while Rubratoxin B was detected only in the juice. These findings provide basic information for evaluating and preventing the risk of contamination by mycotoxins in the citrus fresh fruit supply chain and juice industry.

Phytophthora pseudocryptogea, P. nicotianae and P. multivora Associated to Cycas revoluta: First Report Worldwide.

A dieback was observed on three-year-old pot-grown plants of Cycas revoluta in Sicily (Italy). Symptoms, including stunting, yellowing and blight of the leaf crown, root rot and internal browning and decay of the basal stem, closely resembled the Phytophthora root and crown rot syndrome, common in other ornamentals. Isolations from rotten stem and roots, using a selective medium, and from rhizosphere soil of symptomatic plants, using leaf baiting, yielded three Phytophthora species, P. multivora, P. nicotianae and P. pseudocryptogea, were obtained. Isolates were identified by both morphological characters and DNA barcoding analysis, using three gene regions: ITS, ß-tub and COI. Phytophthora pseudocryptogea was the sole species isolated directly from the stem and roots. The pathogenicity of the isolates of the three Phytophthora species was tested on one-year-old potted plants of C. revoluta, using both stem inoculation by wounding, and root inoculation through infested soil. Phytophthora pseudocryptogea was the most virulent and, like P. nicotianae, reproduced all the symptoms of natural infections, while P. multivora was the least virulent and induced solely very mild symptoms. Phytophthora pseudocryptogea was identified as the causal agent of the decline of C. revoluta, as it was re-isolated from both the roots and stems of artificially infected symptomatic plants, thus fulfilling Koch's postulates.

Fusarium and Neocosmospora Species Associated with Rot of Cactaceae and Other Succulent Plants.

Infections by Fusarium and Fusarium-like species on cacti and other succulent plants cause the syndrome known as Fusarium dry rot and soft rot. There are only few records of Fusarium species as pathogens of cacti and other succulent plants from Iran. The objective of this study was the identification and characterization of fusarioid species recovered from ornamental succulents in Shiraz County, Iran. Three fusarioid species, including F. oxysporum, F. proliferatum, and Neocosmospora falciformis (formerly F. falciforme), were recovered from 29 diverse species of cacti and other succulents with symptoms of Fusarium dry rot and soft rot. The three fungal species were identified on the basis of morphological characters and the phylogenetic analysis of the translation elongation factor1-α (tef1) nuclear gene. The F. oxysporum isolates were identified as F. oxysporum f. sp. opuntiarum. The pathogenicity of the three fusarioid species was tested on a range of economically important ornamental succulents, mostly in the Cactaceae family. The three species showed a broad host spectrum and induced different types of symptoms on inoculated plants, including soft and dry rot, chlorosis, necrotic spots, wilt, drying, root and crown rot. This is the first report of N. falciformis as a pathogen of succulent plants worldwide.

Stilbocrea banihashemiana sp. nov. a New Fungal Pathogen Causing Stem Cankers and Twig Dieback of Fruit Trees.

Stem cankers and twig dieback were the most serious disease of fig (Ficus carica) and loquat (Eriobotrya japonica) noticed in a survey of fruit tree orchards in the Fars Province, Iran. Isolates of Bionectriaceae were consistently recovered from symptomatic fig and loquat trees. Phylogenetic analyses of multiple nuclear loci, internal transcribed spacer regions (ITS) of rDNA, RNA polymerase II subunit 2 (rpb2), and translation elongation factor 1-α (tef1), combined with morphological observations, revealed that isolates could be referred to a still unknown taxon, which was formally described as Stilbocrea banihashemiana sp. nov. Phylogenetically, isolates from fig and loquat trees clustered in a well-supported monophyletic group within the Stilbocrea clade of Bionectriaceae, closely related to S. walteri. Stilbocrea banihashemiana sp. nov. was characterized by the lack of stilbella-like asexual structure in both natural substrates and pure cultures and produced two morphologically distinct types of conidia, globose and cylindrical, formed on short and long simple phialides. In pathogenicity tests, S. banihashemiana sp. nov. induced stem cankers in both fig and loquat, wood discoloration in fig and twig dieback in loquat. Pathogenicity tests also showed that the potential host range of this novel pathogen includes other economically relevant horticultural trees.

Phytophthora × cambivora as a Major Factor Inciting the Decline of European Beech in a Stand within the Southernmost Limit of Its Natural Range in Europe.

The objective of this study was to investigate the role of the oomycete Phytophthora× cambivora in the decline affecting European beech (Fagus sylvatica) in the Nebrodi Regional Park (Sicily, southern Italy). In a survey of a beech forest stand in the heart of the park, Phytophthora× cambivora was the sole Phytophthora species recovered from the rhizosphere soil and fine roots of trees. Both A1 and A2 mating type isolates were found. Direct isolation from the stem bark of trees showing severe decline symptoms and bleeding stem cankers yielded exclusively P. gonapodyides, usually considered as an opportunistic pathogen. The mean inoculum density of P.× cambivora in the rhizosphere soil, as determined using the soil dilution plating method and expressed in terms of colony forming units (cfus) per gm of soil, the isolation frequency using leaf baiting, and the percentage of infected fibrous roots from 20 randomly selected beech trees with severe decline symptoms (50 to 100 foliage transparency classes) were 31.7 cfus, 80%, and 48.6%, respectively. These were significantly higher than the corresponding mean values of 20 asymptomatic or slightly declining trees, suggesting P.× cambivora is a major factor responsible for the decline in the surveyed stand.

DNA Metabarcoding and Isolation by Baiting Complement Each Other in Revealing Phytophthora Diversity in Anthropized and Natural Ecosystems.

Isolation techniques supplemented by sequencing of DNA from axenic cultures have provided a robust methodology for the study of Phytophthora communities in agricultural and natural ecosystems. Recently, metabarcoding approaches have emerged as new paradigms for the detection of Phytophthora species in environmental samples. In this study, Illumina DNA metabarcoding and a conventional leaf baiting isolation technique were compared to unravel the variability of Phytophthora communities in different environments. Overall, 39 rhizosphere soil samples from a natural, a semi-natural and a horticultural small-scale ecosystem, respectively, were processed by both baiting and metabarcoding. Using both detection techniques, 28 out of 39 samples tested positive for Phytophthora. Overall, 1,406,613 Phytophthora internal transcribed spacer 1 (ITS1) sequences and 155 Phytophthora isolates were obtained, which grouped into 21 taxa, five retrieved exclusively by baiting (P. bilorbang; P. cryptogea; P. gonapodyides; P. parvispora and P. pseudocryptogea), 12 exclusively by metabarcoding (P. asparagi; P. occultans; P. psycrophila; P. syringae; P. aleatoria/P. cactorum; P. castanetorum/P. quercina; P. iranica-like; P. unknown sp. 1; P. unknown sp. 2; P. unknown sp. 3; P. unknown sp. 4; P. unknown sp. 5) and four with both techniques (P. citrophthora, P. multivora, P. nicotianae and P. plurivora). Both techniques complemented each other in describing the variability of Phytophthora communities from natural and managed ecosystems and revealing the presence of rare or undescribed Phytophthora taxa.

Epiphytic Yeasts and Bacteria as Candidate Biocontrol Agents of Green and Blue Molds of Citrus Fruits.

Overall, 180 yeasts and bacteria isolated from the peel of citrus fruits were screened for their in vitro antagonistic activity against Penicillium digitatum and P. italicum, causative agents of green and blue mold of citrus fruits, respectively. Two yeast and three bacterial isolates were selected for their inhibitory activity on mycelium growth. Based on the phylogenetic analysis of 16S rDNA and ITS rDNA sequences, the yeast isolates were identified as Candida oleophila and Debaryomyces hansenii while the bacterial isolates were identified as Bacillus amyloliquefaciens, B. pumilus and B. subtilis. All five selected isolates significantly reduced the incidence of decay incited by P. digitatum and P. italicum on 'Valencia' orange and 'Eureka' lemon fruits. Moreover, they were effective in preventing natural infections of green and blue mold of fruits stored at 4 °C. Treatments with antagonistic yeasts and bacteria did not negatively affect the quality and shelf life of fruits. The antagonistic efficacy of the five isolates depended on multiple modes of action, including the ability to form biofilms and produce antifungal lipopeptides, lytic enzymes and volatile compounds. The selected isolates are promising as biocontrol agents of postharvest green and blue molds of citrus fruits.

A super absorbent polymer containing copper to control Plenodomus tracheiphilus the causative agent of mal secco disease of lemon.

The aim of this study was to determine the effectiveness of a Super absorbent polymer (SAP) containing copper (SAP-Cu) in controlling mal secco disease (MSD) of lemon caused by the fungus Plenodomus tracheiphilus. Super absorbent polymer containing copper was characterized by atomic absorption spectrometry (AAS) and UV-VIS spectroscopy. In vitro tests were performed to determine the inhibitory effects of SAP-Cu against the pathogen on both potato-dextrose-agar medium and naturally infected lemon cuttings. Super absorbent polymer was able to absorb up to about 200 and 30 times its weight of ionized water and copper (II) sulfate solution (Cu2+ ions at the concentration 236 mM), respectively. The distribution of copper released on twigs after 24 h of contact with SAP-Cu was determined by secondary ion mass spectrometry with time-of-flight analyzer (ToF-SIMS). Super absorbent polymer containing copper significantly inhibited the viability of P. tracheiphilus in lemon twigs. Overall, the results of this study showed that the SAP could be a suitable carrier of antifungal compounds.

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolarik) in Infected Walnut.

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

Bot Gummosis of Lemon (Citrus × limon) Caused by Neofusicoccum parvum.

Neofusicoccum parvum, in the family Botryosphaeriaceae, was identified as the causal agent of bot gummosis of lemon (Citrus × limon) trees, in the two major lemon-producing regions in Italy. Gummy cankers on trunk and scaffold branches of mature trees were the most typical disease symptoms. Neofusicoccum parvum was the sole fungus constantly and consistently isolated from the canker bark of symptomatic lemon trees. It was identified on the basis of morphological characters and the phylogenetic analysis of three loci, i.e., the internal transcribed spacer of nuclear ribosomal DNA (ITS) as well as the translation elongation factor 1-alpha (TEF1) and ß-tubulin (TUB2) genes. The pathogenicity of N. parvum was demonstrated by wound inoculating two lemon cultivars, 'Femminello 2kr' and 'Monachello', as well as citrange (C. sinensis × Poncirus trifoliata) 'Carrizo' rootstock. In artificial inoculations, the fungus was very aggressive on lemons and weakly virulent on citrange, consistently with symptoms observed in the field as a consequence of natural infections. This is the first report of N. parvum, both in a wide and in a strict taxonomic sense, as a pathogen of lemon in Italy.