Etiology of Septoria Leaf Spot of Pistachio in Southern Spain.

Septoria leaf spot (SLS) is the most prevalent disease of pistachio (Pistacia vera L.) in Spain. To elucidate its etiology, 22 samples of pistachio leaves showing SLS symptoms were collected mainly from 1993 to 2018 across southern Spain. Affected leaves from terebinth (P. terebinthus) were also collected for comparative purposes. Six Septoria-like isolates were recovered from pistachio leaves. They were identified as S. pistaciarum by sequencing internal transcribed spacers, partial RNA polymerase II second largest subunit locus, and 28S ribosomal RNA genes. The phenotypic characteristics of conidia and colonies were evaluated, confirming the identity of S. pistaciarum. Conidia were solitary, hyaline, and straight to curved. Large differences in length were observed between conidia from leaf samples, with those from terebinth being slightly larger than those from pistachio. Colonies showed slow mycelial growth on potato dextrose agar (PDA). The effect of temperature on conidial germination and mycelial growth was evaluated in vitro on PDA. For both characters, the optimum temperature was approximately 19 to 20°C. Eight culture media were tested, with oatmeal agar and Spezieller Nährstoffarmer agar showing the highest mycelial growth and pistachio leaf agar (PLA) showing the highest sporulation. A specific culture medium integrating lyophilized-powdered pistachio leaves into diluted PDA improved sporulation compared with PLA. Pathogenicity tests were conducted by inoculating detached and in planta pistachio and terebinth leaflets with conidial suspensions. Typical symptoms of SLS and cirri of S. pistaciarum developed at 10 and 21 days after inoculation, respectively, in both hosts. To our knowledge, this is the first report of S. pistaciarum causing SLS in pistachio and terebinth in Spain.

Etiology of Branch Dieback and Shoot Blight of English Walnut Caused by Botryosphaeriaceae and Diaporthe Species in Southern Spain.

English walnut (Juglans regia L.) is considered an economically important fruit crop worldwide. In Spain, little attention has been given to walnut diseases owing to the minor economic importance of the walnut crop in the country until recently. In 2017, typical symptoms of branch dieback and shoot blight of English walnut were observed in southern Spain. From 2017 to 2018, 10 commercial walnut orchards showing disease symptoms were surveyed. Botryosphaeriaceae and Diaporthe fungi were consistently isolated from affected shoots. Cytospora isolates were also recovered with minor relevance. Representative isolates of each fungal group were characterized based on colony and conidial morphology, optimum growth temperature, and comparison of DNA sequence data from the internal transcribed spacer, elongation factor 1-α, and ß-tubulin genomic areas. Pathogenicity tests were performed on detached and attached shoots and on detached fruit by inoculating them with mycelial plugs. Botryosphaeriaceae and Diaporthe isolates had higher optimum growth temperatures (≈25 to 27°C) than Cytospora sp. (19.5°C). The following species were identified: Botryosphaeriaceae: Botryosphaeria dothidea, Diplodia seriata, Dothiorella sarmentorum, Dothiorella sp., Neofusicoccum mediterraneum, and N. parvum; Diaporthe: Diaporthe neotheicola, Dia. rhusicola, Diaporthe sp., and Phomopsis amygdali; and Cytospora sp. Botryosphaeriaceae isolates were the most aggressive fungi to walnut in all tissues evaluated, followed by Diaporthe isolates and Cytospora sp. N. parvum was the most virulent among the remaining species tested in any of the tissues evaluated, followed by B. dothidea or N. mediterraneum. This work is the first report to identify the fungal species causing this complex disease of English walnut in Spain and Europe.

Water Stress Enhances the Progression of Branch Dieback and Almond Decline under Field Conditions.

Branch dieback and tree decline have been described as a common complex disease worldwide in woody crops, with Botryosphaeriaceae and Diaporthaceae being considered the most frequent fungi associated with the disease symptoms. Their behaviour is still uncertain, since they are considered endophytes becoming pathogenic in weakened hosts when stress conditions, such as water deficiency occur. Therefore, the main goal of this study was to determine if water stress enhances general decline on weakened almond trees subjected to different irrigation treatments under natural field conditions. In parallel, the occurrence of fungal species associated with almond decline was also determined in relation to disease progression by fungal isolation, and morphological and molecular based-methods. The symptoms of branch dieback and general decline were observed over time, mainly in the experimental plots subjected to high water deficiency. Botryosphaeriaceae were the most consistently isolated fungi, and Botryosphaeria dothidea was the most frequent. Collophorina hispanica was the second most frequent species and Diaporthe and Cytospora species were isolated in a low frequency. Most of them were recovered from both asymptomatic and symptomatic trees, with their consistency of isolation increasing with the disease severity. This work reveals the need to elucidate the role of biotic and abiotic factors which increase the rate of infection of fungal trunk pathogens, in order to generate important knowledge on their life cycle.