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.

Multiple Year Influences of the Aflatoxin Biocontrol Product AF-X1 on the A. flavus Communities Associated with Maize Production in Italy.

AF-X1 is a commercial aflatoxin biocontrol product containing the non-aflatoxigenic (AF-) strain of Aspergillus flavus MUCL54911 (VCG IT006), endemic to Italy, as an active ingredient. The present study aimed to evaluate the long-term persistence of VCG IT006 in the treated fields, and the multi-year influence of the biocontrol application on the A. flavus population. Soil samples were collected in 2020 and 2021 from 28 fields located in four provinces in north Italy. A vegetative compatibility analysis was conducted to monitor the occurrence of VCG IT006 on the total of the 399 isolates of A. flavus that were collected. IT006 was present in all the fields, mainly in the fields treated for 1 yr or 2 consecutive yrs (58% and 63%, respectively). The densities of the toxigenic isolates, detected using the aflR gene, were 45% vs. 22% in the untreated and treated fields, respectively. After displacement via the AF- deployment, a variability from 7% to 32% was noticed in the toxigenic isolates. The current findings support the long-term durability of the biocontrol application benefits without deleterious effects on each fungal population. Nevertheless, based on the current results, as well as on previous studies, the yearly applications of AF-X1 to Italian commercial maize fields should continue.

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.

Does Storage under Gene Bank Conditions Affect Seed Germination and Seedling Growth? The Case of Senecio morisii (Asteraceae), a Vascular Plant Exclusive to Sardinian Water Meadows.

Understanding seed viability under long-term storage conditions provides basic and useful information to investigate the effectiveness of seed banking. Besides the germination success, seedling establishment is also an important requirement, and a decisive step to ensure plant propagation. We used comparative data of germination, seedling growth, and survival percentage between fresh and 10-years-stored seeds of Senecio morisii, a narrow endemic and vulnerable species of Sardinia (Italy), in order to evaluate if differences exist in these traits between fresh and 10-years-stored seeds. Stored seeds showed higher germination percentages than fresh ones, whereas seedling growth and survival did not present significant differences between them, except for seedling growth in plants produced from seeds germinated at 25 °C. This study allowed us to assess if seeds of S. morisii were able to germinate under controlled conditions, and if they maintained their viability and germination capacity for at least 10 years of long-term storage in the seed bank. In addition, the high seedling survival detected in both fresh and stored seeds suggests that stored seeds of S. morisii can be used to support reinforcement or reintroduction actions when fresh materials are not available.