First Report of Gray Leaf Spot Caused by Pyricularia oryzae on Common Millet in Italy.

Common millet (Panicum miliaceum L.) is a niche crop cultivated in Italy for its high nutritional and gluten-free characteristics. These aspects combined with its sustainability and adaptability to paddy soils make it relevant in crop rotation with rice in some organic farms in Northwestern Italy. In September 2021, in a farm producing organic rice located in Rovasenda (VC), Italy (GPS: 45°54'82"N, 8°.31'96"E), plants of millet (var. San Marino), sown on July 1st and almost at physiological maturity, were observed with gray leaf spot symptoms with an incidence of 50% in a 12.5 ha field cultivated in a yearly rotation with rice. Oval shape lesions (between 1 and 5 cm long and 0.5 to1 cm wide) with a light gray colored centers and dark reddish-brown edges were observed on the length of the leaf blade. Gray colonies producing conidia morphologically like Pyricularia developed from 140 symptomatic leaf fragments with a 2% frequency. Monosporic cultures were obtained from five colonies and were grown on Potato Dextrose Agar for morphological and molecular identification. The colonies showed gray, fluffy mycelium, with a smooth margin, with a radius of 28 to 30 mm after 10 days at 28°C. The isolates produced hyaline, pyriform, and 2-septate conidia 17.9 to 33.5 µm (average 25.7 µm) long and 8.3 to 11.7 µm (average 10. µm) wide (n=50) on PDA. Conidiophores were solitary, erect, straight, or curved, septate, and measured 80.2 to 221.3 µm (average 150.7 µm) long and 3.6 to 5.8 µm (average 4.7 µm) wide (n=50). The molecular identification of one single-conidia isolate was confirmed by PCR and sequence comparison of the region of the transposon Pot2 (Harmon et al., 2003). BLASTn searches of GenBank using transposon Pot2 (ON843711) representative isolate 21-03-C, revealed 99.79% identity to P. oryzae (Syn. Magnaporthe oryzae) isolate MD1 (MK608664). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile water at a concentration of 1×105 CFU/ml. Twenty seeds of P. miliaceum were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 28°C under a 12 hours day/night light cycle. Diseased leaves of 10 plants were inoculated by aspersion of 10 ml of the conidial suspension of isolate 21-03-C 2 weeks after the sowing (Harmon et al.; 2003). Plants were incubated in a near moisture-saturated environment in plastic bags at 28°C for 72h, after which symptoms were visually assessed. Ten control plants, inoculated with 10 ml of sterile distilled water remained healthy. P. oryzae was reisolated from leaves showing oblong, whitish lesions, surrounded by light brown margins and identified by resequencing of transposon Pot2. P. oryzae has been already reported on millet in South Korea as causal agent of blast (Klaubauf et al.; 2014). To our knowledge, this is the first report of P. oryzae as a pathogen of millet in Italy and in Europe. Further studies should be conducted to characterize these millet isolates by assessing their virulence on Oryza sativa and the implications in the adoption of P. miliaceum in rotation with rice, to prevent possible carry over of the pathogen on the main crop.

Sequencing of non-virulent strains of Fusarium fujikuroi reveals genes putatively involved in bakanae disease of rice.

Bakanae, one of the most important diseases of rice, is caused by the fungal pathogen Fusarium fujikuroi. The elongation of internodes is the most common symptom induced by the pathogen, and it is related to the production of gibberellins. Despite this, the pathogenicity mechanism of F. fujikuroi is still not completely clear, and there are some strains inducing stunting instead of elongation. Even if there are relatively many genomes of F. fujikuroi strains available in online databases, none of them belongs to an isolate of proven non-virulence, and therefore there has been no comparative genomics study conducted between virulent and non-virulent strains. In the present work, the genomes of non-virulent strain SG4 and scarcely virulent strain C2S were compared to the ones of 12 available virulent isolates. Genes present in the majority of available virulent strains, but not in the non-virulent one, underwent functional annotation with multiple tools, and their expression level during rice infection was checked using pre-existing data. Nine genes putatively related to pathogenicity in F. fujikuroi were identified throughout comparative and functional analyses. Among these, many are involved in the degradation of plant cell wall, which is poorly studied in F. fujikuroi-rice interactions. Three of them were validated through qPCR, showing higher expression in the virulent strain and low to no expression in the low virulent and non virulent strains during rice infection. This work helps to clarify the mechanisms of pathogenicity of F. fujikuroi on rice.

First report of Fusarium commune causing root and crown rot on maize in Italy.

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 62,587,469 t, with an area that covers 628,801 ha, concentrated in northern Italy (ISTAT 2020). Fusarium species are associated with root and crown rot causing failures in crop establishment under high soil moisture. In 2019 maize seedlings collected in a farm located in San Zenone degli Ezzelini (VI, Italy) showed root and crown rot symptoms with browning of the stem tissues, wilting of the seedling, and collapsing due to the rotting tissues at the base of the stem. The incidence of diseased plants was approximately 15%. Seedlings were cleaned thoroughly from soil residues under tap water. Portions (about 3-5 mm) of tissue from roots and crowns of the diseased plants were cut and surface disinfected with a water solution of NaClO at 0.5% for 2 minutes and rinsed in sterile H20. The tissue fragments were plated on Potato Dextrose Agar (PDA) amended with 50 mg/l of streptomycin sulfate and incubated for 48-72 hours at 25oC. Over the 80 tissue fragments plated, 5% were identified as Fusarium verticillioides, 60% as Fusarium spp., 35% developed saprophytes. Fusarium spp. isolates that showed morphological characteristics not belonging to known pathogenic species on maize were selected and used for further investigation while species belonging to F. oxysporum were discarded. Single conidia of the Fusarium spp. colonies were cultured on PDA and Carnation Leaf Agar (CLA) for pathogenicity tests, morphological and molecular identification. The colonies showed white to pink, abundant, densely floccose to fluffy aerial mycelium. Colony reverse showed light violet pigmentation, in rings on PDA. On CLA the isolates produced slightly curved macronidia with 3 septa 28.1 - 65.5 µm long and 2.8-6.3 µm wide (n=50). Microconidia were cylindrical, aseptate, 4.5 -14.0 µm long and 1.5-3.9 µm wide (n=50). Spherical clamydospores were 8.8 ± 2.5 µm size (n=30), produced singly or in pairs on the mycelium, according to the description by Skovgaard et al. (2003) for F. commune. The identity of two single-conidia strains was confirmed by sequence comparison of the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (O'Donnell et al. 2010). BLASTn searches of GenBank, and Fusarium-ID database, using the partial tef-1α (MW419921, MW419922) and rpb2 (MW419923, MW419924) sequences of representative isolate DB19lug07 and DB19lug20, revealed 99% identity for tef-1α and 100% identity to F. commune NRRL 28387(AF246832, AF250560). Pathogenicity tests were carried out by suspending conidia from a 10-days old culture on PDA in sterile H2O to 5×104 CFU/ml. Fifty seeds were immersed in 50 ml of the conidial suspension of each isolate for 24 hours and in sterile water (Koch et al. 2020). The seeds were drained, dried at room temperature, and sown in trays filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 25°C and RH of 80-85% for 14 days with 12 hours photoperiod. Seedlings were extracted from the substrate, washed under tap water, and observed for the presence of root and crown rots like the symptoms observed on the seedlings collected in the field. Control seedlings were healthy and F. commune was reisolated from the symptomatic ones and identified by resequencing of tef-1α gene. F. commune has been already reported on maize (Xi et al. 2019) and other plant species, like soybean (Ellis et al. 2013), sugarcane (Wang et al. 2018), potato (Osawa et al. 2020), indicating that some attention must be paid in crop rotation and residue management strategies. To our knowledge this is the first report of F. commune as a pathogen of maize in Italy. References Ellis M L et al. 2013. Plant Disease, 97, doi: 10.1094/PDIS-07-12-0644-PDN. ISTAT. 2020. http://dati.istat.it/Index.aspx?QueryId=33702. Accessed December 28, 2020. Koch, E. et al. 2020. Journal of Plant Diseases and Protection. 127, 883-893 doi: 10.1007/s41348-020-00350-w O'Donnell K et al. 2010. J. Clin. Microbiol. 48:3708. https://doi.org/10.1128/JCM.00989-10 Osawa H et al. 2020. Journal of General Plant Pathology, doi.org/10.1007/s10327-020-00969-5. Skovgaard K 2003. Mycologia, 95:4, 630-636, DOI: 10.1080/15572536.2004.11833067. Wang J et al. 2018. Plant Disease, 102, doi/10.1094/PDIS-07-17-1011-PDN Xi K et al. 2019. Plant Disease, 103, doi/10.1094/PDIS-09-18-1674-PDN.

Detection of Diverse Maize Chlorotic Mottle Virus Isolates in Maize Seed.

Maize chlorotic mottle virus (MCMV) has driven the emergence of maize lethal necrosis worldwide, where it threatens maize production in areas of East Africa, South America, and Asia. It is thought that MCMV transmission through seed may be important for introduction of the virus in new regions. Identification of infested seed lots is critical for preventing the spread of MCMV through seed. Although methods for detecting MCMV in leaf tissue are available, diagnostic methods for its detection in seed lots are lacking. In this study, ELISA, RT-PCR, and RT-qPCR were adapted for detection of MCMV in maize seed. Purified virions of MCMV isolates from Kansas, Mexico, and Kenya were then used to determine the virus detection thresholds for each diagnostic assay. No substantial differences in response were detected among the isolates in any of the three assays. The RT-PCR and a SYBR Green-based RT-qPCR assays were >3,000 times more sensitive than commercial ELISA for MCMV detection. For ELISA using seed extracts, selection of positive and negative controls was critical, most likely because of relatively high backgrounds. Use of seed soak solutions in ELISA detected MCMV with similar sensitivity to seed extracts, produced minimal background, and required substantially less labor. ELISA and RT-PCR were both effective for detecting MCMV in seed lots from Hawaii and Kenya, with ELISA providing a reliable and inexpensive diagnostic assay that could be implemented routinely in seed testing facilities.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Presence of Powdery Mildew Caused by Erysiphe corylacearum on Hazelnut (Corylus avellana) in Italy.

Hazelnut (Corylus avellana) is widely grown in Italy, which is the second largest producer worldwide with 132,700 tonnes harvested from 78,593 hectares (FAOSTAT, 2018 ). Powdery mildew caused by Phyllactinia guttata has been reported in Italy and in other European countries, but recently in Austria, Switzerland and in central Europe a new species was discovered (Voglmayr et al., 2020; Beenken, 2020). During summer 2020, in Villar Fioccardo (Torino province, Piedmont, Italy) on hazelnut (cv. 'Tonda Gentile') growing on the edges of private gardens and parks, an extensive colonization of the adaxial side of the leaves with white powdery mycelium covering more than 80% of the surface was observed. Also, the abaxial side of the leaves showed the scattered presence of powdery, white, and thin mycelium. The powdery fungal pathogen collected from leaves had amphigenous, hyaline, branched, septate 1.5 to 3.7 µm wide mycelium; lobed, solitary hyphal appressoria; vertically elevated above the mycelium 53 to 82 µm long and 5 to 12 µm wide conidiophores (n = 30); hyaline, ellipsoid, ovoid to doliform conidia, solitary on conidiophores, 21 to 36 µm long, 15 to 21 µm wide (average 28 to 18 µm) (n = 50). Chasmothecia appeared in late September 2020 and they were spherical, single or in groups, 83 to 138 (average 100) µm in diameter (n = 50); 7 to 15 aseptate appendages were straight, sometimes flexuous, 55 to 111 (average 73) µm long (n = 50), with four to five times dichotomous branched apexes and recurved tips. In each chasmothecium, there were three to five ellipsoid, ovoid to subglobose asci with a length of 41 to 60 µm and a width of 28 to 56 µm (average 52 to 44 µm) (n = 30). Asci contained four to eight ascospores, 15 to 26 µm long and 10 to 17 µm wide (average 19 to 12 µm) (n =50). Mycelia were carefully scraped from the leaves with a scalpel and DNA was extracted by using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany). Partial rDNA internal transcribed spacer region (ITS) of two isolates (DB20SET01, DB20SET01) was amplified using specific primers PMITS1/PMITS2 (Cunnington et al. 2003) and sequenced. Obtained sequences were deposited in GenBank (Accession Nos. MW045425, MW045426). BLAST analysis of the obtained 749-bp fragments showed 100% identity to ITS rDNA sequences of Erysiphe corylacearum from Switzerland (MN822721) and Azerbaijan (LC270863). One-year-old plants of C. avellane cv. Tonda Gentile were artificially inocuated by dusting conidia from infected leaves. Inoculated plants were incubated under controlled conditions at 23°C ± 1 and 70 to 80% relative humidity. Typical symptoms (white bloom) appeared on the upper surface of the leaves at 8 to 10 days after inoculation. No symptoms were found on control plants treated with sterile water. The fungus isolated from inoculated leaves was morphologically identical to the original isolates from diseased plants collected from Villar Fioccardo. Erysiphe corylacearum causes a new and aggressive form of powdery mildew. Since the first observation in north-eastern Turkey in 2013, it has spread rapidly throughout the Black Sea region, causing significant economic losses (Sezer et al., 2017). It has also been reported in Iran, Azerbaijan, and Ukraine (Arzanlou et al. 2018; Heluta et al., 2018). The disease has been observed sporadically in Piedmont, Italy, during summer 2020 (Regione Piemonte & Agrion, 2020) in some hazelnut growing areas, but presently, doesn't appear to impact yield. This is the first report of E. corylacearum, causing an aggressive powdery mildew on hazelnut in Italy, and as such, may more severely affect hazelnut groves in Italy and cause considerable yield losses. Literature cited Arzanlou M et al. 2018. Forest Pathology, 48:e12450. https://doi.org/10.1111/efp.12450. Beenken L et al. 2020. New Disease Reports 41, 11. http://dx.doi.org/10.5197/j.2044-0588.2020.041.011. Cunnington JH et al. 2003. Australasian Plant Pathology, 32, 421-428. Food and Agriculture Organization (FAO). 2018. http://www.fao.org/faostat/en/#home Heluta V.P. et al.2019. Ukrainian Botanical Journal, 2019, 76(3), 252-259. Regione Piemonte SFR & Agrion. 2020. https://www.regione.piemonte.it/web/sites/default/files/media/documenti/2020-10/mal_bianco_nocciolo_da_erysiphe_corylacearum.pdf Sezer AD et al. 2017. Phytoparasitica, 45, 577-581. Voglmayr H et al. 2020. New Disease Reports, 42, 14 http://dx.doi.org/10.5197/j.2044-0588.2020.042.014.

Diversity and Pathogenicity of Fusarium Species Associated with Stalk and Crown Rot in Maize in Northern Italy.

The genus Fusarium includes several agronomically important and toxin-producing species that are distributed worldwide and can cause a wide range of diseases. Crown and stalk rot and grain infections are among the most severe symptoms that Fusarium spp. can cause in maize. Disease development usually occurs during germination, but it may also affect the later phases of plant growth. The purpose of this study was to investigate the diversity and pathogenicity of 41 isolates recovered from symptomatic seedlings collected in Northern Italy and seeds of five different geographical origins in 2019 and 2020. The pathogenicity was tested and confirmed in 23 isolates causing rotting in maize seedlings, with disease indexes from 20% to 90%. A multilocus phylogeny analysis based on four genomic loci (tef1-α, rpb2, calm and tub2) was performed on 23 representative isolates. Representative isolates were identified as species belonging to three species complexes (SC), including Fusarium verticillioides and F. annulatum in the F. fujikuroi SC. Fusarium commune was identified in the F. nisikadoi SC, and three different lineages were found in the Fusarium oxysporum SC. This study reports F. annulatum and two lineages of the Fusarium oxysporum SC as maize pathogens for the first time in Italy.

Assessment of microbial biocontrol agent (BCA) viability to mechanical and thermal stress by simulating spray application conditions.

BACKGROUND: In order to improve the biological control agent (BCA) efficacy, stress factors threatening the viability of microorganisms during spray application need to be determined. The effect of spray mixture temperature and exposure time on Trichoderma harzianum T 22 and Bacillus amyloliquefaciens QST713 viability were tested. Concurrently the combined effect of mechanical and thermal stress effect on BCA viability were tested at two initial spray mixture temperatures (14 and 25 °C) by simulating a spray application using airblast sprayers featured by different tank capacity and a spray liquid circuit (without and with hydraulic agitation system). To assess the BCA microorganism viability, spray mixture samples were collected at time intervals along trials and plated to count the colony forming units (CFU). RESULTS: The critical temperature threshold that inhibited BCA viability was 35 °C with 30 min of exposure. The sprayer type, the initial temperature of the spray mixture and the temperature increment during the trials significantly decreased the number of CFU recovered. When simulating a spray application, the spray mixture temperature increase rate was determined mainly by the residual amount of spray mixture in the tank. Even if the tank capacity does not substantially affect the final temperature reached by the spray mixture, the higher residual spray mixture in bigger tanks can expose the BCAs for a longer time to critical temperatures. CONCLUSIONS: Experimental trials allowed us to identify the effect of factors affecting the viability of tested BCAs, providing information about the actual chance to guarantee the biological efficacy of BCA treatments. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Phytosanitary Interventions for Safe Global Germplasm Exchange and the Prevention of Transboundary Pest Spread: The Role of CGIAR Germplasm Health Units.

The inherent ability of seeds (orthodox, intermediate, and recalcitrant seeds and vegetative propagules) to serve as carriers of pests and pathogens (hereafter referred to as pests) and the risk of transboundary spread along with the seed movement present a high-risk factor for international germplasm distribution activities. Quarantine and phytosanitary procedures have been established by many countries around the world to minimize seed-borne pest spread by screening export and import consignments of germplasm. The effectiveness of these time-consuming and cost-intensive procedures depends on the knowledge of pest distribution, availability of diagnostic tools for seed health testing, qualified operators, procedures for inspection, and seed phytosanitation. This review describes a unique multidisciplinary approach used by the CGIAR Germplasm Health Units (GHUs) in ensuring phytosanitary protection for the safe conservation and global movement of germplasm from the 11 CGIAR genebanks and breeding programs that acquire and distribute germplasm to and from all parts of the world for agricultural research and food security. We also present the challenges, lessons learned, and recommendations stemming from the experience of GHUs, which collaborate with the national quarantine systems to export and distribute about 100,000 germplasm samples annually to partners located in about 90 to 100 countries. Furthermore, we describe how GHUs adjust their procedures to stay in alignment with evolving phytosanitary regulations and pest risk scenarios. In conclusion, we state the benefits of globally coordinated phytosanitary networks for the prevention of the intercontinental spread of pests that are transmissible through plant propagation materials.

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.

Maize lethal necrosis (MLN): Efforts toward containing the spread and impact of a devastating transboundary disease in sub-Saharan Africa.

Maize lethal necrosis (MLN), a complex viral disease, emerged as a serious threat to maize production and the livelihoods of smallholders in eastern Africa since 2011, primarily due to the introduction of maize chlorotic mottle virus (MCMV). The International Maize and Wheat Improvement Center (CIMMYT), in close partnership with national and international partners, implemented a multi-disciplinary and multi-institutional strategy to curb the spread of MLN in sub-Saharan Africa, and mitigate the impact of the disease. The strategy revolved around a) intensive germplasm screening and fast-tracked development and deployment of MLN-tolerant/resistant maize hybrids in Africa-adapted genetic backgrounds; b) optimizing the diagnostic protocols for MLN-causing viruses, especially MCMV, and capacity building of relevant public and private sector institutions on MLN diagnostics and management; c) MLN monitoring and surveillance across sub-Saharan Africa in collaboration with national plant protection organizations (NPPOs); d) partnership with the private seed sector for production and exchange of MLN pathogen-free commercial maize seed; and e) awareness creation among relevant stakeholders about MLN management, including engagement with policy makers. The review concludes by highlighting the need to keep continuous vigil against MLN-causing viruses, and preventing any further spread of the disease to the major maize-growing countries that have not yet reported MLN in sub-Saharan Africa.

Monitoring the threat of unintentional transgene flow into maize gene banks and breeding materials

The use of transgenic crops is steadily increasing around the world, led by soybean (based on total area) and maize (in terms of total number of countries). Transgenic maize is grown in at least 17 countries across four continents: Africa, America, Asia and Europe. The comprehensive global spread of transgenic maize has significant implications for organizations involved in germplasm conservation and genetic enhancement; particularly as some countries require a GMO-free declaration when receiving shipments of maize germplasm. This article describes the protocol used by the International Maize and Wheat Improvement Center (CIMMYT) for monitoring unintentional transgene flow in maize genebank and breeding plots. The protocol is based on polymerase chain reaction (PCR) markers for detecting specific recombinant DNA sequences in bulked samples collected from sentinel plots. To date, no unintentional transgene flow has been detected in CIMMYT fields of maize genebank accessions or breeding materials.