Twenty-five almond cultivars were assessed for susceptibility to Diaporthe amygdali, causal agent of twig canker and shoot blight disease. In laboratory experiments, growing twigs were inoculated with four D. amygdali isolates. Moreover, growing shoots of almond cultivars grafted onto INRA ‘GF-677’ rootstock were used in four-year field inoculations with one D. amygdali isolate. In both type of experiments, inoculum consisted of agar plugs with mycelium, which were inserted underneath the bark and the lesion lengths caused by the fungus were measured. Necrotic lesions were observed in the inoculated almond cultivars both in laboratory and field tests, confirming the susceptibility of all the evaluated cultivars to all the inoculated isolates of D. amygdali. Cultivars were grouped as susceptible or very susceptible according to a cluster analysis. The relationship between some agronomic traits and cultivar susceptibility was also investigated. Blooming and ripening times were found relevant variables to explain cultivars performance related to D. amygdali susceptibility. Late and very late blooming, and early and medium ripening cultivars were highly susceptible to D. amygdali. Our results may provide valuable information that could assist in ongoing breeding programs of this crop and additionally in the selection of cultivars for new almond plantations.
From 2018 to 2020, surveys of oomycetes associated with root and crown rot of almond (Prunus dulcis) were conducted on diseased young almond trees in commercial orchards and nurseries in six provinces of Spain. A total of 104 oomycete isolates were obtained from plant and soil samples, which h were identified by sequencing the internal transcribed spacer (ITS) region of the ribosomal DNA. Diverse species belonging to the genera Globisporangium, Phytophthora, Phytopythium and Pythium were found, Phytopythium vexans and Phytophthora niederhauserii being the most frequent. The pathogenicity of these two species to one-year-old almond seedlings of ‘Garnem’ (P. dulcis × P. persica) rootstock was studied. All seedlings inoculated with Pp. vexans and Ph. niederhauserii isolates showed severe symptoms at the late stage of the pathogenicity test (defoliation, wilting and dieback) and several plants died. Some isolates of Ph. niederhauserii significantly reduced the dry weight of the roots compared with the control, but this effect was not observed in seedlings inoculated with Pp. vexans. These results provide new information about the oomycete species present in almond crops in Spain and highlight the importance of carrying out frequent phytosanitary surveys for a better knowledge of potential risks posed by these soil-borne pathogens.
Nectarine (Prunus persica (L.) Batsch var. nucipersica (Suckow) C. K. Schneid.) is a fruit crop widely cultivated throughout the Mediterranean basin. In Spain, it is mainly grown in eastern regions of the country. In March 2018, 5-year-old nectarine trees showing twig canker symptoms were observed after a rainy spring period in a 0.5 ha orchard located at Alaior, Menorca island (Spain). Cankers were frequent on affected trees (approximately, 80% of the total trees), thus leading to shoot blight. Ten twig segments of one-year old wood with cankers were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution and rinsed twice in sterile distilled water. Small pieces (2 mm) of affected tissues were taken from the margin of the cankers and plated on potato dextrose agar (PDA) supplemented with 0.5 g/L of streptomycin sulphate (PDAS). The plates were then incubated at 25 ºC in the dark for 7 to 10 d. Actively growing colonies were first hyphal-tipped and then transferred to PDA and 2% water agar supplemented with sterile pine needles and incubated at 21-22ºC under a 12h/12h near UV / darkness cycle during 21 d (León et al. 2020). Colonies were white at first, becoming light cream, with visible solitary and aggregate pycnidia at maturity. Alpha conidia were aseptate, fusiform, hyaline, multi-guttulated (mean ± SD = 7.4 ± 0.7 × 2.8 ± 0.4 µm, n = 100). Beta and gamma conidia were not observed. The morphological and cultural characteristics of the isolates were congruent with those of Diaporthe spp. (Gomes et al. 2013). The ITS1-5.8S-ITS2 (ITS) region and fragments of β-tubulin (tub2), the translation elongation factor 1-alpha (tef1-α) gene regions, histone H3 (his3) and calmodulin (cal) genes of representative isolate DAL-59 were amplified and sequenced (Santos et al. 2017). The BLASTn analysis revealed 100% similarity with sequences of D. mediterranea (Synonym D. amygdali) (Hilário et al. 2021) isolate DAL-34 from almond (ITS: MT007489, tub2: MT006686, tef1-α: MT006989, his3: MT007095 and cal: MT006761). Sequences of isolate DAL-59 were deposited in GenBank Database (ITS: MT007491, tub2: MT006688, tef1-α: MT006991, his3: MT007097 and cal: MT006763). Pathogenicity tests were conducted using one-year-old potted plants of nectarine cv. Boreal, which were inoculated with isolate DAL-59. In each plant, a 3 mm wound was made in the center of the main branch (about 30 cm length) with a scalpel. Colonized agar plugs with 3 mm diameter, which were obtained from active 10-day-old colonies growing on PDA, were inserted underneath the epidermis and the wounds sealed with Parafilm. Inoculated plants were incubated in a growth chamber at 23 ºC with 12 h of light per day. Controls were inoculated with uncolonized PDA plugs. There were twelve plants per treatment, which were arranged in a completely randomized design. Five days after inoculation necrosis development was observed in the area of inoculation. Wilting and twig blight symptoms over the lesion occurred 3-wk after inoculation and pycnidia were detected, while the controls remained asymptomatic. Diaporthe amygdali was re-isolated from symptomatic tissues and identified as described above to satisfy Koch’s postulates. To our knowledge, this is the first report of D. amygdali causing twig canker and shoot blight disease on nectarine in Spain.
Diseases caused by soil-borne oomycetes are a limiting factor for the cultivation of Prunus spp., which makes the choice of a suitable rootstock a key factor. The objective of this study was to evaluate the pathogenicity of 12 oomycete species belonging to the genera Globisporangium, Phytophthora (Ph.) and Phytopythium (Pp.) to three Prunus hybrid rootstocks: 'Garnem', 'GF-677' and 'Rootpac-40'. These three rootstocks are widely used to grow stone fruit and almond in the Mediterranean Basin. Pathogenicity tests were conducted using 15 oomycete isolates and 1-year-old rootstock seedlings. Ninety days after inoculation, disease symptoms were evaluated on a severity scale, and the Area Under the Disease Progression Curve and the survival probability of the inoculated seedlings were calculated. Moreover, root dry weight was recorded. All the isolates included in the pathogenicity tests were pathogenic on the rootstock seedlings and were re-isolated from root lesions. Large differences in virulence were detected among the different oomycete species and isolates of Ph. niederhauserii for each rootstock. Phytophthora multivora and Pp. helicoides were generally the most virulent species. The results of the present research offer substantial contribution to increase our knowledge about the pathogenicity of several oomycete species that are frequently isolated in Prunus orchards, and the potential risks that they pose for Prunus spp. crops.
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