First Report of Alternaria alternata and Fusarium spp. Causing Brown Apical Necrosis in Walnut Fruit in Southern Chile

This is the first report of Alternaria alternata and Fusarium spp. causing brown apical necrosis (BAN) in walnut fruit in southern Chile. English walnut (Juglans regia L.) is the second most widely grown fruit in Chile. The bacterial pathogen Xanthomonas arboricola pv. juglandis affects walnut production in Chile and has been associated with apical necrosis symptom and premature fruit drop; this research focused on determining if fungal pathogens were associated with this damage. The presence of BAN in commercial walnut orchards in southern Chile reveals the need for improved phytosanitary programs to control this disease.

Computational Identification and Comparative Analysis of Conserved miRNAs and Their Putative Target Genes in the Juglans regia and J. microcarpa Genomes

MicroRNAs (miRNAs) are important factors for the post-transcriptional regulation of protein-coding genes in plants and animals. They are discovered either by sequencing small RNAs or computationally. We employed a sequence-homology-based computational approach to identify conserved miRNAs and their target genes in Persian (English) walnut, Juglans regia, and its North American wild relative, J. microcarpa. A total of 119 miRNA precursors (pre-miRNAs) were detected in the J. regia genome and 121 in the J. microcarpa genome and miRNA target genes were predicted and their functional annotations were performed in both genomes. In the J. regia genome, 325 different genes were targets; 87.08% were regulated by transcript cleavage and 12.92% by translation repression. In the J. microcarpa genome, 316 different genes were targets; 88.92% were regulated by transcript cleavage and 11.08% were regulated by translation repression. Totals of 1.3% and 2.0% of all resistance gene analogues (RGA) and 2.7% and 2.6% of all transcription factors (TFs) were regulated by miRNAs in the J. regia and J. microcarpa genomes, respectively. Juglans genomes evolved by a whole genome duplication (WGD) and consist of eight pairs of fractionated homoeologous chromosomes. Within each pair, the chromosome that has more genes with greater average transcription also harbors more pre-miRNAs and more target genes than its homoeologue. While only minor differences were detected in pre-miRNAs between the J. regia and J. microcarpa genomes, about one-third of the pre-miRNA loci were not conserved between homoeologous chromosome within each genome. Pre-miRNA and their corresponding target genes showed a tendency to be collocated within a subgenome.

Fungal Trunk Pathogens Associated With Juglans regia in the Czech Republic

Juglans regia L. (English walnut) trees with cankers and dieback symptoms were observed in two regions in the Czech Republic. Isolations were made from diseased branches. In total, 138 fungal isolates representing 10 fungal species were obtained from wood samples and identified based on morphological characteristics and molecular methods: Cadophora novi-eboraci, Cadophora spadicis, Cryptovalsa ampelina, Diaporthe eres, Diplodia seriata, Dothiorella omnivora, Eutypa lata, Eutypella sp., Peroneutypa scoparia, and Phaeoacremonium sicilianum. Pathogenicity tests conducted under field conditions with all species using the mycelium-plug method indicated that Eutypa lata and Cadophora spp. were highly virulent to woody stems of walnut. This is the first study to detect and identify fungal trunk pathogens associated with diseased walnut trees in Europe.

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 β-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.

Interaction Between Diaporthe rhusicola and Neofusicoccum mediterraneum Causing Branch Dieback and Fruit Blight of English Walnut in California, and the Effect of Pruning Wounds on the Infection

Botryosphaeriaceae and Diaporthaceae species are the causal agents of branch dieback of English walnut in California. In this study, the effects of the interaction between Neofusicoccum mediterraneum and Diaporthe rhusicola were evaluated in vitro by using mycelial plugs or spore suspensions and in vivo by inoculating shoots and epicarps (hulls) of walnut. Single inoculations of each species and different coinfection treatments were performed under laboratory or field conditions. The influence of shoot age and susceptibility of bark or pith tissues to N. mediterraneum and D. rhusicola infection after pruning was also evaluated. In in vitro experiments, spore germination of D. rhusicola was significantly (P < 0.0001) reduced in the presence of N. mediterraneum spores. When D. rhusicola was inoculated at 4 days before N. mediterraneum, a delay in lesion development in shoots and hulls was observed compared with the other two interaction treatments. One- to 2-year-old shoots were more susceptible to infection and colonization by N. mediterraneum than 3- to 4-year-old shoots. In young shoots, inoculation in the pith tissue resulted in longer lesions than those observed on shoots inoculated in the bark. No significant differences were observed between the development of internal or external necrosis and the age of the shoots, or the susceptibility of bark and pith to D. rhusicola infection. This information is essential to better understanding the complex situation of this walnut disease toward developing control management strategies.

Characterization of Oomycete Species Associated With Root and Crown Rot of English Walnut in Chile

English (Persian) walnut (Juglans regia) trees affected by root and crown rot were surveyed in five regions of central Chile between 2015 and 2017. In each region, nine orchards, ranging from 1 to 21 years old, were randomly selected and inspected for incidence and severity of tree decline associated with crown and root rot. Soil and symptomatic crown and root tissues were collected and cultured in P5ARP semiselective medium to isolate potential oomycete pathogens, which were identified through morphology and molecularly using ITS sequences in the rDNA gene and beta tubulin gene. The most frequently isolated species was Phytophthora cinnamomi. Pathogenicity tests were conducted with representative oomycete isolates. P. cinnamomi, P. citrophthora, and Pythium ultimum were all pathogenic in J. regia. Nevertheless, only P. cinnamomi and P. citrophthora were pathogenic to English walnut. Py. ultimum caused limited levels of root damage to English walnut seedlings. Our research indicates that as the Chilean walnut industry has expanded, so have walnut crown and root rots induced by oomycetes.