First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, ß-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

Use of hydroponics culture to assess nutrient supply by treated wastewater.

The use of treated wastewater for irrigation is increasing, especially in those areas where water resources are limited. Treated wastewaters contain nutrients that are useful for plant growth and help to reduce fertilizers needs. Nutrient content of these waters depends on the treatment system. Nutrient supply by a treated wastewater from a conventional treatment plant (CWW) and a lagooned wastewater from the campus of the University of Balearic Islands (LWW) was tested in an experiment in hydroponics conditions. Half-strength Hoagland nutrient solution (HNS) was used as a control. Barley (Hordeum vulgare L.) seedlings were grown in 4 L containers filled with the three types of water. Four weeks after planting, barley was harvested and root and shoot biomass was measured. N, P, K, Ca, Mg, Na and Fe contents were determined in both tissues and heavy metal concentrations were analysed in shoots. N, P and K concentrations were lower in LWW than in CWW, while HNS had the highest nutrient concentration. Dry weight barley production was reduced in CWW and LWW treatments to 49% and 17%, respectively, comparing to HNS. However, to a lesser extent, reduction was found in shoot and root N content. Treated wastewater increased Na content in shoots and roots of barley and Ca and Cr content in shoots. However, heavy metals content was lower than toxic levels in all the cases. Although treated wastewater is an interesting water resource, additional fertilization is needed to maintain a high productivity in barley seedlings.

Chemical properties and biological activity in soils of Mallorca following twenty years of treated wastewater irrigation.

On the Mediterranean island of Mallorca, the use of secondary-treated municipal wastewater in irrigation was introduced with the construction of the first wastewater treatment plants in the 1970s. In this study, the chemical properties and biological activity of 21 arable soils, irrigated for more than 20 years with secondary-treated wastewater, were tested in order to assess their quality. Soil quality was evaluated by measuring cation exchange capacity, pH, calcium carbonate equivalent, soil organic matter, total nitrogen, available phosphorus, water-soluble organic carbon, soil microbial biomass, soil basal respiration, and the activities of the enzymes dehydrogenase, ß-glucosidase and alkaline phosphatase. No negative effects of the irrigation treatment were observed on the measured soil parameters. Indeed, soil water-soluble organic carbon, soil microbial biomass and ß-glucosidase and alkaline phosphatase activities increased under treated wastewater irrigation. Biological activity of soils irrigated with treated wastewater was affected mainly by soil organic matter content. Although the typical crop management of alfalfa, and other forage crops associated with treated wastewater irrigation, may have contributed to the increase of these parameters, the results suggest that irrigation with treated wastewater is a strategy with many benefits to agricultural land management.