Symptomatic Trees Research Articles

Transcriptomic analyses reveal physiological changes in sweet orange roots affected by citrus blight

BackgroundCitrus blight is a very important progressive decline disease of commercial citrus. The etiology is unknown, although the disease can be transmitted by root grafts, suggesting a viral etiology. Diagnosis is made by demonstrating physical blockage of xylem cells that prevents the movement of water. This test was used to identify symptomatic trees from four commercial groves in Florida. Total RNA extracts of phloem-enriched scaffold root tissues were prepared from seven trees that failed to take up water and from one healthy tree. These RNA extracts were used for transcriptomic analyses using paired end RNA-Seq from an Illumina 2500 system. The expression of transcripts annotated as polyprotein of citrus endogenous pararetrovirus were estimated by both RT-qPCR and RNA-Seq.ResultsTranscripts from seven RNA-Seq libraries from trees affected by citrus blight were compared to a control tree. 129–148 million RNA fragments (two paired-end reads/fragment) were generated per library and were mapped to the sweet orange reference genome. In response to citrus blight stress, genes encoding aquaporins, proteins with water channel activity and several cellulose synthase genes were down-regulated, whereas genes involved in lignin and glucosinolate biosynthesis were up-regulated. Transcripts encoding proteins in pathways of carbohydrate metabolism, nucleotide synthesis, signaling, hormone metabolism, secondary metabolism, transport, and biotic stress pathways were overwhelmingly down regulated in all libraries.ConclusionReduced water intake and xylem plugging were observed in the trees tested and the changes in their transcriptome were analyzed. Plants adapted to reduced water flow by regulating primary and secondary metabolism, nuclear transport and hormone associated pathways. The patterns of energy generation, transcription, translation and protein degradation were consistent with irreversible decline. The down regulation of cellulose synthase transcripts and up regulation of transcripts related to lignin production likely lead to an imbalance in the pathways leading to wood formation, and may lead to the blockage of the xylem vessels seen as the cardinal symptom of citrus blight. Transcripts of a pararetrovirus were elevated in the transcriptome of roots used in this study.

Differential selection pressure exerted by root rot disease on the microbial communities in the rhizosphere of avocado (Persea americana Mill.)

AbstractDespite the high economic impact of root rot disease, knowledge regarding the rhizosphere microflora of avocado trees affected by root rot is limited. Metagenomics was applied to identify the difference in the rhizosphere microflora of avocado trees with and without visible symptoms of root rot. Approximately, 446,970 common gene catalogues differed between them, confirming that root rot affected the bacterial and fungal communities in the rhizosphere. The proportion of bacterial genera, namely, Labilithrix, Sorangium, Sandaracinus and Pedosphaera showed a decrease while Phenylobacterium, Rhizomicrobium, Candidatus Solibacter and Silvibacterium genera were increased by root rot. The proportion of fungal genera, namely, Pseudogymnoascus, Moelleriella, Mortierella, Lepidopterella, Babjeviella, Lachancea, Macrophomina, Pneumocystis, Sugiyamaella and Cyphellophora showed an increase while Cryptococcus, Verticillium, Bipolaris, Pyrenochaeta, Rhizophagus, Cenococcum and Neonectria genera were inhibited by root rot. Moreover, the proportion of the top 10 bacteria in the rhizosphere of symptomatic trees was significantly higher, and that of the top 10 fungi was significantly lower, compared to the asymptomatic trees. Principal component analysis based on abundance analysis and function prediction showed that in symptomatic trees, the bacterial community was more concentrated, while the fungal community was more dispersed. The differences in the responses of bacterial and fungal genera suggested that the pathogenic fungi exert varying selection pressure on the microflora. Moreover, root rot affected the metabolism of carbohydrates, lipids and amino acids in bacteria, and the global and overview maps, carbon metabolism and processing of genetic and environmental information in fungi, which might result in differential selection pressure.

Phytophthora root and collar rots in fruit orchards in the Czech Republic

A recent outbreak of Phytophthora diseases in fruit orchards was identified in the Czech Republic. The diseased trees showed characteristic symptoms including yellowing, wilting and sparse foliage, decreased yields, root and collar rot, and withering and dying of trees. In some orchards up to 10–15, and rarely up to 55%, of trees died. In total, 387 symptomatic trees of nine species from 44 fruit orchards, 16 samples of irrigation water from four orchards and 35 samples of nursery stock, were surveyed in 2012–2018. Oomycetes were recovered from 50.6 % of sampled trees in orchards, from 71.4 % of shipments of ex vitro-produced nursery plants, and from 93.8 % irrigation water samples. Seventeen Phytophthora species and 13 Pythium sensu lato species were recovered. The most frequent species in orchards were Phytophthora cactorum, Phytophthora plurivora and Phytopythium vexans. The most frequent species in nursery stock were P. cactorum, Phy. vexans and Globisporangium intermedium, and Pythium helicoides, Phytophthora lacustris and Pythium litorale were the most frequently found in irrigation water. The most frequent oomycete species recovered from nursery stock were also frequently isolated from orchards and planting material. The pathogenicity of 11 Phytophthora species was assessed in vitro in two frequently used rootstocks – ´St. Julien´ (plum) and ´M26´ (apple). This revealed that the less known and relatively rarely isolated species (P. gregata, P. chlamydospora × amnicola, P. inundata, P. lacustris and P. sansomeana) were more virulent than the more frequently occurring species P. cactorum and P. plurivora, and could pose potential threats to fruit orchards in the future. The increase of Phytophthora in fruit orchards is probably related to the trading and planting of infected nursery stock, to climate change and water stress, and to dismissal of the problem of Phytophthora diseases by stakeholders and inappropriate management. Future investigation should focus on the development of effective disease management, including assessment and selection of rootstocks for resistance.

Fourier-transform infrared (FT-IR) spectroscopy analysis discriminates asymptomatic and symptomatic Norway spruce trees

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 – 912 cm−1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 – 994 cm−1 and 1104 – 994 cm−1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

Phytoplasmas associated with declining of hazelnut (Corylus avellana) in Slovenia

Hazelnut (Corylus avellana) is cultivated on 118 ha and ranks eighth in Slovenian fruit growing production, representing 2.8% of the total area of fruit plantations in the country. However, decline of some of the trees appeared in 2012 in two plantations located in eastern Slovenia. Together these orchards cover 5 ha, with around 1600 trees planted 12 to 15 years ago. By October 2018, ~12% of these trees had died, and an additional 12% showed decay symptoms. The dead and dying trees were scattered throughout both orchards, with no apparent pattern. The most affected cultivar was ‘Istrska dolgoplodna leska’. Using molecular diagnostic methods, we showed infection of symptomatic trees with three unrelated phytoplasmas: ‘Candidatus Phytoplasma fragariae’, of the 16SrXII-E phytoplasma subgroup, and phytoplasma of the 16SrV and 16SrIX groups. In 2018, the presence of ‘Ca. P. fragariae’ and/or phytoplasma of 16SrV group were confirmed in decayed hazelnut trees in eastern, north-eastern, central, south-eastern and western Slovenia. ‘Ca. P. fragariae’ has also been detected in a forest in south-western Slovenia, for Acer campestre, Carpinus betulus, Crataegus laevigata, Fraxinus ornus and Quercus petraea. All infected forest trees showed unusual dense proliferation of sprouts from roots and/or trunks. Molecular characterisations of partial 16S rRNA, secY, map and ribosomal protein genetic locus of hazelnut 16SrV phytoplasma isolates show that they are identical to isolates that can cause grapevine flavescence dorée disease. Here, the results of our recent study and the open questions on this burning issue for hazelnut production are presented.

First Report of Leaf Spot on Oil Palm Caused by Phyllosticta capitalensis in Malaysia

HomePlant DiseaseVol. 104, No. 1First Report of Leaf Spot on Oil Palm Caused by Phyllosticta capitalensis in Malaysia PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Leaf Spot on Oil Palm Caused by Phyllosticta capitalensis in MalaysiaA. Nasehi, H. Sathyapriya, and M. Y. WongA. Nasehihttp://orcid.org/0000-0002-5926-0742Institute of Plantation Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiaSearch for more papers by this author, H. SathyapriyaInstitute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiaSearch for more papers by this author, and M. Y. Wong†Corresponding author: M. Y. Wong; E-mail Address: muiyun@upm.edu.myhttp://orcid.org/0000-0002-6944-4860Institute of Plantation Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiaDepartment of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiaSearch for more papers by this author AffiliationsAuthors and Affiliations A. Nasehi1 H. Sathyapriya2 M. Y. Wong1 3 † 1Institute of Plantation Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Published Online:6 Nov 2019https://doi.org/10.1094/PDIS-06-19-1232-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Oil palm (Elaeis guineensis Jacq.) is the most economically important crop in Malaysia and the world’s highest yielding oil crop. In March 2018, irregular spots with gray centers, dark brown edges, and chlorotic halos were observed on the leaves of oil palm trees of Tenera genotype planted at Block A, University Agriculture Park, Universiti Putra Malaysia, Malaysia. The area of production is 8 ha of conventional farming with temperature and humidity ranging from 33 to 34°C and 30 to 70%, respectively. There were a total of 1,192 oil palm trees in the area analyzed, and the disease incidence was estimated to be approximately 20%. Twelve leaf sections (5 mm2) of four diseased leaves from four different symptomatic oil palm trees were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed twice with distilled water, dried on sterilized tissue paper, plated on potato dextrose agar (PDA) plates, and incubated at 27 ± 1°C for 14 days in the dark. A total of 12 single-spore isolates were obtained from all sampled leaf tissues. The single colonies of all fungal isolates were olivaceous greenish to olivaceous black with an irregular light olive margin on PDA. Conidia were obpyriform or nearly elliptic, unicellular, with a hyaline, unstable apical appendage, 5 to 12 µm long. The size of conidia was 10 to 15 μm in length and 6 to 8 μm in width. These morphological characters were consistent with the original description of Phyllosticta capitalensis (Glienke et al. 2011; Hennings 1908). For fungal identification to species level, the internal transcribed spacer region (ITS) of the representative isolate UPM-Ph1 was amplified utilizing the universal primers ITS5/ITS4 (White et al. 1990) and then sequenced. The 620-bp ITS sequence was deposited in GenBank under accession number MH699964. The phylogenetic analysis confirmed that the isolate UPM-Ph1 belonged to P. capitalensis, and it shares 100% homology with the ITS sequence of the reference pathogenic P. capitalensis strain CPC18884 (GenBank JF261465), which was isolated from the host Stanhopea graveolens in Brazil, as reported by Glienke et al. (2011). Pathogenicity testing was conducted by artificial inoculation of healthy oil palm (Tenera GH 500 series) leaves with isolate UPM-Ph1 under the conditions of 9-h day/15-h night cycle and using LED white light. Four leaves were wound inoculated in three parts with a sterile scalpel and pipetting a 10-µl droplet of a conidial suspension (106 conidia/ml) on each wound. Another four leaves were wound inoculated with sterilized distilled water and served as a control. All inoculated leaves and control leaves were incubated in a chamber with 98% relative humidity at 27 ± 1°C. After 7 days, typical lesions identical to those observed in the field appeared on the inoculated leaves but not on control leaves. P. capitalensis was successfully reisolated only from the inoculated leaves and identified by morphological observations. The pathogenicity test was repeated, and the same typical lesions were observed. P. capitalensis has been reported as a weak plant pathogen that causes various diseases in plants (Wikee et al. 2013a, 2013b). Unsuccessful reisolation of the fungus from the control leaves confirmed that the control plants were not already latently infected with the pathogen. To our knowledge, this is the first report of P. capitalensis causing leaf spot disease of oil palm in Malaysia. This pathogen is capable of affecting the health and yield of oil palm trees in the plantations substantially if no control measure is in place. Therefore, further investigation is needed to determine the distribution of P. capitalensis in oil palm plantations in Malaysia.The author(s) declare no conflict of interest.

Xanthomonas perforans: a tomato and pepper pathogen associated with bacterial blight and dieback of Eucalyptus pellita seedlings in Indonesia

Leaf and shoot blight, often accompanied by die-back symptoms, on Eucalyptus species, hybrids and clones have been reported from a number of countries. More than one bacterial species has been found to cause these symptoms. In this study, a leaf disease of E. pellita in Indonesia was investigated. The disease was found primarily on nursery plants and young trees that recovered within the first year of growth. Leaf samples were collected from symptomatic trees, and isolations consistently yielded a Xanthomonas sp. Sequencing of the 16S rRNA gene region and multilocus sequence analysis (MLSA) was performed on 19 of the 61 Xanthomonas isolates obtained. In the MLSA, four genes, namely, dnaK, fyuA, gyrB and rpoD, were sequenced and the isolates were identified as X. perforans. Four representative isolates, at a concentration of 106 CFU/ml, were leaf-infiltrated and spray-inoculated on to E. pellita, tomato and pepper seedlings. The type isolate of X. perforans was included in the pathogenicity trials as a positive control. All four isolates of X. perforans, inclusive of the type isolate, induced bacterial spot symptoms on tomato and pepper seedlings. They also caused water-soaked lesions on the leaves of E. pellita seedlings, characteristic of the symptoms observed in the field. This is the first report of X. perforans infecting leaves of a woody host.

Leaf litter decomposition of sweet chestnut is affected more by oomycte infection of trees than by water temperature

Abstract Riparian forests are subjected to multiple disturbances, such as tree diseases caused by invasive pathogens, whose consequences on stream functioning are unknown. We assessed the impact of Phytophthora cinnamomi infection, and interactions with temperature, on microbial decomposition of Castanea sativa leaves. Leaves from healthy, symptomatic and highly symptomatic trees were incubated in the laboratory at 13 and 18 °C for 64 d. Infection significantly increased polyphenolic concentration and leaf toughness, reducing leaf decomposition and microbial respiration rates irrespective of temperature. Aquatic hyphomycete communities differed significantly in leaves from highly symptomatic trees. Fungal biomass was highest at 18 °C, irrespective of tree health status. None of the parameters were influenced by the tree health status × temperature interaction, suggesting that temperature rise may not synergistically increase the cross-ecosystem effects caused by P. cinnamomi in streams where litter decomposition is microbial-driven. Infection by P. cinnamomi alters the nutritional quality of leaves affecting the functioning of aquatic ecosystems.

Bacillus pumilus and Stenotrophomonas maltophilia as two potentially causative agents involved in Persian oak decline in Zagros forests (Iran)

AbstractThe oak decline is known as one of the most destructive complex diseases causing high economic losses around the world, especially in Iran. The main objective of the present study was to investigate the possible role of bacteria as causative agents of oak decline in the Zagros forests of Iran. To do this, stem, root and leaf samples were taken from symptomatic Persian oak trees (Quercus brantii) in different zones of Zagros forests (Ilam Province, Iran). From 150 bacterial isolates, 20 showed pathogenicity against Geranium seedlings. Among 20 hypersensitivity test positive strains, four strains showed pathogenicity against oak saplings. Based on morphological and 16S rRNA gene sequence analysis, three strains were identified as Bacillus pumilus and one strain as non‐sporulating Gram‐negative Stenotrophomonas maltophilia. Pathogenicity studies of different B. pumilus and S. maltophilia strains revealed that they have potential to cause the disease in oak saplings and symptoms of disorder in Persian oak trees. To our knowledge, there are no previous records of B. pumilus and S. maltophilia causing decline on Fagaceous trees like Q. brantii. More detailed field and molecular studies are required to confirm the absolute role of such bacteria in occurrence of oak decline in Zagros forests.

First Report of Neoscytalidium dimidiatum Causing Black Canker and Root Rot of Walnut in Turkey

Walnut (Juglans regia L.) is a commercially important crop with about 87,670 ha grown in Turkey, the fourth largest producer worldwide. During summer 2018, unfamiliar and serious decline symptoms associated with stem and branch cankers were observed in walnut trees in two orchards in Sanliurfa Province, Southeastern Anatolia Region. In a 3-year-old orchard of 450 trees (cv. Chandler) in Kayseri, Siverek District, about 62% of trees had deep, large, and black cankers in the trunks and on main branches, drastically reduced tree vigor, and yellowing, wilting, and browning of the leaves, followed by leaf drop. In a 12-year-old orchard with 30 trees (cv. Kaman), in Yaylak, Bozova District, 40% of trees had similar symptoms. Symptoms were first noticed in trunks. Bark cracks, cankers in trunks, and dark discoloration or a black sooty, dusty mass of fungal spores under peeling bark on the trunk and main branches were the most striking symptoms. Deep cankers expanded longitudinally, extending along trunks and into the phloem of the trees, which consequently died, and occasionally laterally extending into the sapwood. Declining trees had dry root rot symptoms, with black necrosis on the surface, and under the cracked and coarse bark extending from the taproot and some large roots to the crown, and black rot and loss of fine roots. Samples were taken from under the bark of trunk cankers, crown tissues, and root tissues of five trees per orchard. The sections of samples were surface sterilized in 2% NaOCl for 2 min and rinsed twice in sterile distilled water, blotted dry with sterilized filter paper, placed on PDA, and incubated at 28°C. A fungus with identical cultural characteristics was consistently isolated. On PDA, the mycelium growth was white and then turned black with age. Rapidly growing colonies (average, 40 mm/day) produced mycelia that disarticulated into 0- to 1-septate, cylindrical-truncate or rod-shaped, thick-walled, hyaline to brown arthroconidia occurring singly or in arthric chains and averaged 9.6 ± 3.7 × 5.5 ± 1.6 μm with a length/width ratio of 1.79. These characteristics were consistent with the description of the arthric synanamorph of Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). The rDNA internal transcribed spacer (ITS) and large subunit (LSU) genes of two single conidial isolates (one from under bark tissues; one from root tissues) were amplified with ITS6/ITS4 and NL1/NL4 primer pairs, respectively, and the amplicons sequenced. The ITS and LSU sequences deposited in GenBank (ITS, MK430050 and MK430051; LSU, MK430056 and MK430057) had 100% identity with many sequences of N. dimidiatum, including recently available ones (Turkolmez et al. 2019; ITS, MH114591; LSU, MH114592), in line with the morphological features. Pathogenicity tests were performed on 20 replicates of 1-year-old container-grown J. regia ‘Chandler’ seedlings with a N. dimidiatum isolate by removing a 5-mm-diameter bark plug from the stem with a cork borer and placing an equivalent-sized 7-day-old culture disc of N. dimidiatum onto the exposed vascular cambium. The wound was covered with wet, autoclaved cotton wool and sealed with Parafilm. Treated plants were maintained in a growth chamber with a 16/8-h light/dark photoperiod at 26°C and 55% RH and watered on demand. Within 3 weeks, average lesion lengths under the bark of all inoculated stems were >150 mm, and the lesions were covered with a black sooty layer. Leaf blight was also observed, similar to naturally affected trees. Control plants remained healthy. N. dimidiatum was reisolated only from lesions of inoculated stems, fulfilling Koch’s postulates. In Turkey, N. dimidiatum has been reported as a destructive new blight of tomato (Turkolmez et al. 2019) and canker, shoot blight, and root rot of pistachio (Dervis et al. 2019). This is the first report of N. dimidiatum infection of walnuts in Turkey. By the end of 2018, all symptomatic trees had died. Recent occurrence of this severe disease suggests it could have a significant potential for limiting walnut production.

Phytophthora species associated with dieback of sweet chestnut in Western Turkey

AbstractSweet chestnut (Castanea sativa) is an important tree species in the Marmara and Aegean regions of Turkey as these two regions produce the great majority of edible nuts, especially those used for marron glacé production. Chestnut forests and orchards in these regions showing severe dieback symptoms not associated with chestnut blight were investigated to determine the role of Phytophthora spp. in the decline syndrome. Soil samples were collected from around 108 symptomatic chestnut trees at 29 sites and Phytophthora spp. isolated using soil baiting technique and selective medium. Species isolated were identified by cultural characteristics and ITS sequencing. Phytophthora x cambivora was the dominant species detected in 13 sites, followed by P. cinnamomi (5 sites), P. plurivora (3 sites) and P. cryptogea (1 site). Phytophthora x cambivora was present in both regions, while P. cinnamomi was found only in the Marmara region in coastal areas around Istanbul. When inoculated at the stem bases of 3‐year‐old chestnut saplings, P. cinnamomi produced significantly longer necrotic lesions (7.8–12.0 cm) than P. x cambivora (2.6–6.3 cm) by 12 days after inoculation. Phytophthora plurivora was the least aggressive species causing only small lesions. Phytophthora cryptogea, which represents the first record on chestnut in Turkey, produced intermediate sized lesions in between P. x cambivora and P. plurivora. These results indicate that P. x cambivora and in some areas P. cinnamomi play major roles in the observed dieback of sweet chestnut in western Turkey.

First Report of Neofusicoccum parvum Causing Stem Canker and Necrosis of Phoebe sheareri in Zhejiang Province, China

Phoebe sheareri is a popular species established in Zhejiang province of China for timber. During surveys in June 2016, severe canker problems were observed in one plantation of P. sheareri in Kaihua county, Zhejiang. The disease started with black fusiform necrosis (8 to 15 mm long, 3 to 5 mm wide) on the green bark of shoots, and the cankers could be found on branches and even the main stem (with diameter up to 10 cm). These cankers can girdle stems, resulting in the death of plant tissues above. Ten stem sections containing the junction between healthy and necrotic xylem from five symptomatic trees were collected. Sections of symptomatic xylem were surface sterilized in 5% sodium hypochlorite water solution and placed on potato dextrose agar (PDA) at 25°C in the dark. After 3 days, seven isolates were generated by subculturing single hyphal tips onto fresh PDA medium and grown for 7 days in the dark. All seven isolates initially developed white aerial mycelium that later turned dark gray. After 2 weeks, black globular pycnidia could be found. The fusiform hyaline conidia were aseptate, with thin walls, and measured 20.9 ± 2.2 × 5.1 ± 0.4 μm. Three isolates (KHZJZ1 to KHZJZ3, respectively) were selected randomly for molecular characterization. The rDNA internal transcribed spacer (ITS), partial β-tubulin (tub2), and translation elongation factor 1-α (tef1) gene regions were amplified by polymerase chain reaction using the primer pairs ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). Gene sequences of all three fungal isolates were identical. The ITS, tub2, and tef1 sequences were deposited in GenBank (accession nos. MK131330, MK139576, and MK139575, respectively). A local BLASTn search revealed that the ITS, tub2, and tef1 sequences shared 99, 99, and 100% identity, respectively, to corresponding sequences of ex-type isolate Neofusicoccum parvum strain CMW9081 (accession nos. AY236943, AY236917, and AY236888, respectively). Pathogenicity tests were conducted by wounding stems of 2-year-old P. sheareri seedlings with a 5-mm-diameter cork borer. Wounds were inoculated with PDA plugs excised from 7-day-old cultures of the N. parvum isolate (KHZJZ1). Stems inoculated with sterile PDA plugs acted as controls. The inoculated stem wounds were wrapped with Parafilm, and the seedlings were kept under natural conditions grown in the field (18 to 30°C). There were five replicates of each treatment. After 21 days, the N. parvum-inoculated stems had developed black necrotic lesions (11 ± 5 mm long) on the bark, whereas the control plants remained asymptomatic. The fungus was reisolated from diseased plant tissue but not from the controls, thus fulfilling Koch’s postulates. N. parvum has previously been reported as the causal agent of canker and die-back of Eucalyptus (Iturritxa et al. 2011), stem blight of blueberry (Koike et al. 2014), and canker of other woody plants (Jayakumar et al. 2011). To our knowledge, this is the first report of N. parvum causing canker of P. sheareri, which is becoming increasingly important in timber plantations in south China.

Development of pathogenicity assay and characterization of Fusarium oxysporum f. sp. elaeidis (FOE) based on Secreted In Xylem genes and EF-1α

The pathogenicity of four Fusarium oxysporum isolates collected from symptomatic oil palm (Elaeis guineensis Jacq.) from Ghana were tested for the first time to develop a new pathogenicity assay for Fusarium oxysporum f. sp. elaeidis (FOE) infection in oil palm seedlings. All four FOE isolates used for pathogenicity assay were pathogenic to oil palm seedlings within a relatively short time compared to other pathogenicity studies, for which infection/symptoms in oil palm seedlings was time consuming. FOE and “presumed-FOE” (i.e. Fusarium isolates collected from symptomatic oil palm trees whose pathogenicity is not confirmed) were characterised based on partial sequences of a housekeeping gene EF-1α and three Secreted In Xylem genes (SIX8, SIX9 and SIX11). All the phylogenetic trees generated for EF-1α, SIX8, SIX9 and SIX11 showed some variation between FOE, and “presumed-FOE”, but could not cluster isolates based on geographical location. Phylogenetic trees for EF-1α and SIX (SIX9 and SIX11) genes clustered both FOE and “presumed-FOE” from FUSARIUM-ID from GenBank, but SIX8 could not.

Characterization of Fusarium and Neocosmospora Species Associated With Crown Rot and Stem Canker of Pistachio Rootstocks in California.

California produces 99.1% of pistachios grown in the United States, and diseases affecting pistachio rootstocks represent a constant challenge to the industry. Field surveys of fungi associated with pistachio rootstocks with symptoms of crown rot and stem canker in three central California counties followed by phylogenetic analyses of translation elongation factor 1-α and second largest subunit of RNA polymerase II gene fragments identified three Fusarium species (Fusarium equiseti, Fusarium oxysporum, and Fusarium proliferatum) and two Neocosmospora species (Neocosmospora falciformis and Neocosmospora solani). F. oxysporum and N. falciformis were the fungal species most frequently recovered from symptomatic pistachio trees. Inoculations of detached twigs of cultivar Kerman pistachio Pioneer Gold I and clonal University of California, Berkeley I (UCBI) rootstocks showed that all five species could colonize pistachio wood and cause vascular discolorations. Pathogenicity tests in potted pistachio trees completed Koch's postulates and confirmed that F. oxysporum, F. proliferatum, N. falciformis, and N. solani were capable of producing rot and discoloration in stems of clonal UCBI rootstocks, the most widely planted pistachio rootstock in California. To our knowledge, this study is the first to present insights into the biodiversity and biology of Fusarium and Neocosmospora species associated with pistachio trees in California.

Genetic diversity of hop stunt viroid from symptomatic and asymptomatic citrus trees in Iran

AbstractHop stunt viroid as the causal agent of cachexia disease has detected from citrus trees in different areas in Iran. Although cachexia has not been reported as a decline disease for citrus trees, it can impair crop quality and reduce plant yields. This study was undertaken to molecularly detect HSVd among different commercial citrus cultivars and determine genetic diversity of this viroid in Mazandaran province of Iran. Sampling was performed from symptomatic and symptomless citrus cultivars in Mazandaran province. HSVd specific primers were used for molecular detection. SSCP and sequencing were applied to assay HSVd genetic diversity. Results showed the detection of HSVd in all symptomatic Satsuma (25 out of 25), Clementine (25 out of 25), sweet lime (20 out of 20) and sweet orange cv. Valencia (7 out of 7), as well as, 31% (14 out of 22), 100% (12 out of 12) and 33% (5 out of 15) of page mandarin, lemon and grapefruit trees, respectively. 10 different HSVd genomes were identified by sequencing the SSCP profiles among which HSVd‐IR1 had the most frequency.

Detection of Huanglongbing disease based on intensity-invariant texture analysis of images in the visible spectrum

Huanglongbing (HLB) is considered one of the most threatening diseases for citrus production and has caused economic damage in many countries around the world. Hence, citrus producers need in-field tools for the early detection of HLB to control its spread. Technological approaches are widely used for in-field HLB detection: algorithms of image analysis have demonstrated their potential to detect HLB in color images automatically. Specifically, texture features have been commonly used for characterizing HLB and other nutrient deficiencies. However, because the systems for HLB detection are intended to operate in-field, illumination of the environment is unlikely to be always the same; thus, texture analysis can be sensitive to the illumination. To overcome this limitation, a method for HLB detection based on intensity-invariant texture analysis is presented in this study. The ranklet transform is used to convert the input image to an intensity-invariant representation, from which common texture features are extracted. A random forest classifier is used to distinguish between distinct classes of citrus leaves, including healthy, nutritionally deficient, and HLB. The experimental results show the robustness of the proposed approach to different types of illumination: the classification performance remains stable independent of the brightness of the input image. An accuracy of about 95% in distinguishing between HLB-infected and HLB-negative classes was achieved, and an accuracy of about 81% in identifying between six classes of citrus leaves. These results reveal the potential of the proposed approach to be implemented within a mobile application that can be used in-field for HLB detection in symptomatic citrus trees.

An Evaluation of Monitoring Surveys of the Quarantine Bacterium Xylella Fastidiosa Performed in Containment and Buffer Areas of Apulia, Southern Italy.

Xylella fastidiosa is a quarantine phytopathogen for the European Plant Protection Organization and currently infects olive trees in the Apulia region (southern Italy). Upon the Implementing Decision of the European Union 2016/764 of May 12, 2016, extensive monitoring surveys were performed on approximately 190 000 ha to ascertain the possible occurrence of X. fastidiosa. The primary objectives of the analysis were to start to collect epidemiological data on X. fastidiosa occurrence in areas far from the initial outbreaks and discuss the results of the pathogen detection. A total of 220 279 olive trees were inspected. Basic information on farm and trees management was obtained. A total of 13 706 olive trees were analyzed through serological and molecular techniques to verify the possible occurrence of the bacterium. The cultivars "Nociara," "Cima di Melfi," and "Cellina di Nardò" showed the highest occurrence of decline symptoms. Tree age appears to be related to the incidence of decline symptoms. Olive trees growing in well-managed soils showed fewer symptoms than trees cultivated in farms where such agronomic techniques are not regularly performed. X. fastidiosa was detected in 2078 samples taken from symptomatic trees and 1653 samples obtained from asymptomatic trees. In 3300 samples taken from symptomatic trees, the bacterium was not detected. Implementation and utilization of reliable in situ detection techniques could increase the number of sampled trees in each plot, thus allowing a more extensive and robust assessment of X. fastidiosa-infected plants in areas where the pathogen inoculums are still low.

Economic implications of a native tree disease, Caliciopsis canker, on the white pine (Pinus strobus) lumber industry in the northeastern United States

In the northeastern United States, eastern white pine (Pinus strobus L.) is a leading species in the forest products industry. The native pathogen Caliciopsis pinea Peck is associated with Caliciopsis canker of white pine, with symptoms including excessive resin production and cankers. This study processed 28.0 m3 of white pine lumber to (i) quantify losses resulting from Caliciopsis canker, (ii) assess how damage varies between Caliciopsis canker symptom severity and thinning, and (iii) quantify economic loss resulting from damage. Caliciopsis canker damage was present in 37% of lumber, yet only 10% was downgraded due to canker damage. Of the downgraded lumber, the vast majority (77%) lost one grade. Additionally, severely symptomatic trees consistently had more damage, and their lumber was more likely to be downgraded than trees with low symptom severity. Caliciopsis canker damage resulted in average revenue losses of 2.3%, yet much of the sampled lumber had other, more significant damage that resulted in downgrade: highly symptomatic trees averaged 63% of the revenue of low or asymptomatic trees. Caliciopsis canker, therefore, can be used as an indicator of poor quality trees. We recommend thinning Caliciopsis canker symptomatic trees to meet low-density stocking guidelines, which may minimize revenue loss while simultaneously minimizing stress to residual stock.

Could tree density play a role in managing Citrus Huanglongbing epidemics?

Citrus Huanglongbing (HLB) management relies on adoption of preventive measures including the use of healthy nursery trees, area-wide control of the vector Diaphorina citri, and eradication of symptomatic trees. Cultural practices including tree density could contribute to HLB management. We investigated the role of tree density on the incidence of HLB using a historical dataset from commercial orchards in Sao Paulo State, Brazil. The HLB incidence from 2013 to 2015 was compared through 495 blocks of different tree ages (3 to 13 years old), tree localization within the farm (edge or non-edge), and tree densities (from 220 to 714 trees·ha−1) according to the tree spacing used. Diaphorina citri was controlled rigorously in all commercial blocks using drench applications of systemic insecticides and foliar sprays of contact insecticides. The results indicated that cumulative HLB incidence was predominantly lower at higher tree densities. In addition to vector control and removal of HLB-symptomatic trees, planting of trees in high densities may reduce the proportions of HLB-infected trees, thereby contributing to improved profitability for farmers in HLB-endemic sites. In particular, this management practice should be applied at the edges of farms.

The Previously Unidentified, Divergent Badnavirus Species Cacao red vein-banding virus is Associated with Cacao Swollen Shoot Disease in Nigeria.

Cacao swollen shoot disease (CSSD) of Theobroma cacao was reported in Nigeria in 1944; however, no badnaviral genome sequences have been found associated with the symptomatic trees. In 2017, leaf samples (n = 18) were collected from cacao trees from Osun and Oyo, Nigeria showing foliar symptoms that included red vein-banding and shoot swelling, and variable secondary mosaic, mottling, and fern-like pattern symptoms. Abutting primers designed around previously determined 500-bp intergenic region sequences were used for polymerase chain reaction (PCR) amplification. Of the 18 samples, 9 yielded an approximately 7,000-bp, apparently genome-size product. The nine genomes were sequenced and found to encode four open reading frames, and to share 86 to 99% nucleotide identity. Pairwise analysis of the Nigerian genomes with 21 previously reported CSSD badnaviruses, at the complete genome and reverse-transcription ribonuclease H (1,230 bp) sequence levels, indicated 71 to 75 and 72 to 76% nucleotide identity, respectively. Phylogenetic analysis of the nine complete genomes indicated that the closest relatives of the divergent Nigerian isolates were previously described West African CSSD badnaviruses. Based on pairwise comparisons and phylogenetic analyses, the Nigerian CSSD isolates constitute a previously unrecognized Badnavirus sp., herein named Cacao red vein-banding virus (CRVBV). Primers designed based on the CRVBV genome sequences amplified a 1,068-bp fragment from 16 of 18 field samples tested by PCR, suggesting the possible existence of additional CRVBV variants.