RESUMO
Almond (Prunus dulcis Mill. D. A. Webb) is considered a major crop in the Mediterranean countries. In Italy, almond orchards cover an area of 54,939 ha, with a yield of 82,146 t, with the cultivation widely distributed across several areas, such as southern regions. During the summer of 2023, 3-year-old almond plants cultivated in a commercial orchard in southern Piedmont (44°32'41.92'' N; 7°57'25.7'' E) showed wilting and sudden decline of the whole plant with an incidence of 15%. Main symptoms were cankers on the trunk characterized by a chocolate-brown discoloration of wood, cambium and even bark and occasional presence of gummy exudate. Symptomatic bark samples were rinsed with water, then blotted dry, and briefly soaked in ethanol for 15 s. Small pieces (5 × 5 mm) from the margin of necrotic areas were cut and placed on potato dextrose agar (PDA) plates amended with streptomycin sulphate (150 µg/ml). After incubation in the dark at 25 °C for 4 days fungal colonies emerging from the plated fragments were subcultured as monohyphal fungal cultures. They were fast-growing, turning from withish to smoky grey or grey-olivaceous after a week. Stromatic conidiomata were produced in pine needle agar, with pycnidia forming hyaline, globose to ellipsoidal, unicellular conidia, ranging in size from 16.9-17.3 µm × 5.4-5.6 µm. The causal agent was preliminary identified as Neofusicoccum sp. based on morphology. Two representative isolates (MTB3, MTC4) were identified by sequencing the ITS region and translation elongation factor 1-alpha (TEF1-α) gene with the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences of the two isolates were deposited in NCBI GenBank with accession numbers PP930646 / PP930647 (ITS), and PP933185 / PP933186 (TEF-1α). BLAST analysis revealed 99% similarity with reference sequences of Neofusicoccum parvum isolate CMW9081. A maximum-likelihood phylogenetic tree was constructed using MEGA7 software based on the combined ITS and TEF1-α dataset. The phylogenetic tree demonstrated that the two isolates grouped with N. parvum in a clade with the ex-type CMW9081. The pathogenicity of both isolates was evaluated on 1-year-old 'Vairo' almond potted plants grafted on GF677 rootstock, to assess Koch's postulate. For the inoculation, 5 mm in diameter mycelium plugs taken from a 7-days-old PDA culture were placed on the surface of fresh wounds made with a sterile cork-borer (5 mm-diameter) on the stem. Control plants were inoculated with sterile PDA plugs. The test was conducted twice. Three blocks of three plants per isolate and control were arranged in a randomized block design. All inoculated plants showed symptoms of shoot blight and cankers 2-weeks after inoculation. Additionally, gumming, pycnidia production and internal wood discoloration developed on the inoculation sites, with lesion length spreading for 16.8 ± 1.6 and 15.4 ± 3.0 cm for MTB3 and MTC4, respectively. Most of the plants died one month after inoculation. Control plants showed no symptoms. The pathogen was consistently reisolated from inoculated plants and the identity was confirmed through TEF-1α sequencing. Thus, results of Koch postulate clearly identify N. parvum as the etiological agent of the observed disease of almond in Piedmont. N. parvum was recently reported in the same area on hazelnut (Waqas et al. 2021) and blueberry (Guarnaccia et al. 2021), and as causal agent of band canker and branch dieback in almond trees in US and Spain (Moral et al. 2019). To our knowledge, this is the first report of N. parvum as the causal agent of almond decline syndrome in Italy. Considering the increasing of almond cultivation in Italy, this disease represents a threat for the future production.
RESUMO
The European "Green Deal" policies are shifting toward more sustainable and environmentally conscious agricultural practices, reducing the use of chemical fertilizer and pesticides. This implies exploring alternative strategies. One promising alternative to improve plant nutrition and reinforce plant defenses is the use of beneficial microorganisms in the rhizosphere, such as "Plant-growth-promoting rhizobacteria and fungi". Despite the great abundance of iron (Fe) in the Earth's crust, its poor solubility in calcareous soil makes Fe deficiency a major agricultural issue worldwide. Among plant promoting microorganisms, the yeast Debaryomyces hansenii has been very recently incorporated, for its ability to induce morphological and physiological key responses to Fe deficiency in plants, under hydroponic culture conditions. The present work takes it a step further and explores the potential of D. hansenii to improve plant nutrition and stimulate growth in cucumber plants grown in calcareous soil, where ferric chlorosis is common. Additionally, the study examines D. hansenii's ability to induce systemic resistance (ISR) through a comparative relative expression study by qRT-PCR of ethylene (ET) biosynthesis (ACO1), or ET signaling (EIN2 and EIN3), and salicylic acid (SA) biosynthesis (PAL)-related genes. The results mark a significant milestone since D. hansenii not only enhances nutrient uptake and stimulates plant growth and flower development but could also amplify induced systemic resistance (ISR). Although there is still much work ahead, these findings make D. hansenii a promising candidate to be used for sustainable and environmentally friendly integrated crop management.