No abstract

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 60,602,320 t, covering 588,597 ha (ISTAT 2021). Trichoderma species are widespread filamentous fungi in soil, well known and studied as biological control agents (Vinale et al., 2008). Seeds of a yellow grain hybrid (class FAO 700, 132 days) were collected in September 2020 from an experimental field located in Carmagnola (TO, Italy: GPS: 44°53'11.0"N 7°40'60.0"E) and tested with blotter test (Warham et al., 1996) to assess their phytosanitary condition. Over the 400 seeds tested, more than 50% showed rotting and development of green mycelium typical of the genus Trichoderma. Due to the high and unexpected percentage of decaying kernels, ten colonies were identified by morphological and molecular methods. Single conidia colonies of one Trichoderma (T5.1) strain were cultured on Potato Dextrose Agar (PDA) for pathogenicity tests, and on PDA and Synthetic Nutrient-Poor Agar (SNA) for morphological and molecular identification. The colonies grown on PDA and SNA showed green, abundant, cottony, and radiating aerial mycelium, and yellow pigmentation on the reverse. Colony radius after 72 h at 30°C was of 60-65 mm on PDA and of 50-55 mm on SNA. The isolates produced one cell conidia 2.8 - 3.8 µm long and 2.1 - 3.6 µm wide (n=50) on SNA. Conidiophores and phialides were lageniform to ampulliform and measured 4.5 – 9.7 µm long and 1.6 – 3.6 µm wide (n=50); the base measure 1.5 – 2.9 µm wide and the supporting cell 1.4 – 2.8 µm wide (n=50). The identity of one single-conidia strain was confirmed by sequence comparison of the internal transcribed spacer (ITS), the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (Oskiera et al., 2015). BLASTn searches of GenBank using ITS (OL691534) the partial tef-1α (OL743117) and rpb2 (OL743116) sequences of the representative isolate T5.1, revealed 100% identity for rpb2 to T. afroharzianum TRS835 (KP009149) and 100% identity for tef-1α to T. afroharzianum Z19 (KR911897). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile H2O to 1×106 CFU/ml. Twenty-five seeds were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 23°C under a seasonal day/night light cycle. Twenty primary ears were inoculated, by injection into the silk channel, with 1 ml of a conidial suspension of strain T5.1 seven days after silk channel emergence (BBCH 65) (Pfordt et al., 2020). Ears were removed four weeks after inoculation and disease severity, reaching up to 75% of the kernels of the twenty cobs, was assessed visually according to the EPPO guidelines (EPPO, 2015). Five control cobs, inoculated with 1 ml of sterile distilled water were healthy. T. afroharzianum was reisolated from kernels showing a green mold developing on their surface and identified by resequencing of tef-1α gene. T. afroharzianum has been already reported on maize in Germany and France as causal agent of ear rot of maize (Pfordt et al. 2020). Although several species of Trichoderma are known to be beneficial microorganisms, our results support other findings that report Trichoderma spp. causing ear rot on maize in tropical and subtropical areas of the world (Munkvold and White, 2016). The potential production of mycotoxins and the losses that can be caused by the pathogen during post-harvest need to be explored. To our knowledge this is the first report of T. afroharzianum as a pathogen of maize in Italy.

No abstract

Hypericum scruglii is an endangered endemic plant of Sardinia and the phloroglucinol compounds identified in this species have been reported to inhibit Human Immunodeficiency Virus activity. Seed banks are a genetic repository that effectively preserve taxa of conservation interest and they hold knowledge about the biology and germination eco-physiology of the taxa they preserve. The main goals of this study were to investigate the germination requirements of H. scruglii, to evaluate the seed viability after eight years of long-term conservation and to suggest an efficient protocol for germination. Seeds stored at -25 °C in the seed bank were tested at temperatures from 5 to 30 °C and 25/10 °C. Base temperature (Tb) and thermal time (θ50) for germination were estimated. H. scruglii seeds germinated over a wide range of temperatures, responding positively to high temperatures and 25/10 °C. Tb was estimated at 4.92 °C, and θ50 was estimated at 198.27 degree days. The high viability detected in seeds of H. scruglii allows us to suggest the use of seeds stored in a seed bank when fresh material for plant propagation is scarce. Our results provide new and useful baseline information for implementing conservation and multiplication strategies for this endangered medicinal plant.

Fusarium fujikuroi, causing bakanae disease, is one of the most important seedborne pathogens of rice, the detection of which is paramount for seed certification and for preventing field infections. Molecular tests—qPCR and loop-mediated isothermal amplification (LAMP)—are replacing the blotter test in seed health procedures, due to higher sensitivity, specificity, fast turnaround results delivery, on-site application and the possibility of quantifying endophytic seed infections. A LAMP test, which had been previously developed with primers designed to target the elongation factor 1-α sequence of F. fujikuroi, was validated according to the international validation standard (EPPO, PM7/98) on thirty-four rice seed lines of different levels of susceptibility to the disease, thus comparing it to the blotter test and with two different DNA extraction procedures. The use of crude extracted DNA provided more sensitive results than the DNA extracted with the commercial kit Omega E.Z.N.A® Plant DNA kit. The results showed that the endophytic infection of F. fujikuroi is essential for the development of the disease in the field and that the minimum amount of the pathogen necessary for the development of the disease corresponds to 4.17 × 104 cells/µL. This study confirms the applicability of the LAMP technique on-site on rice seeds with fast and quantitative detection of the pathogen.