Headspace solid‐phase microextraction gas chromatography–mass spectrometry for the detection of volatile organic compounds released from Ganoderma boninense and oil palm wood

Basal stem rot disease caused by the white‐rot fungus Ganoderma boninense is a major threat to the oil palm industry, and hence, the ability to detect infections at an early stage of development is desired. In this study, a headspace solid‐phase microextraction (HS‐SPME) method coupled with gas chromatography–mass spectrometry (GC‐MS) was employed to analyse the volatile organic compounds (VOCs) released from G. boninense cultures and infected oil palm wood. We examined VOCs released from three types of samples: G. boninense mycelium, oil palm wood and oil palm wood colonized by G. boninense. This preliminary study led to the tentative identification of 57 VOCs, including alcohols, alkanes, volatile acids, ketones, aldehydes, esters, sesquiterpenes and polycyclic aromatic hydrocarbon groups. Aliphatic compounds with eight‐carbon atoms, such as 1‐octen‐3‐ol, 3‐octanone, 1‐octanol and (E)‐2‐octenal, were the most abundant constituents of the Ganoderma samples, whereas furfural and hexanal were the major compounds detected in the oil palm wood samples. Chemometric analyses using cluster heat maps and principal component analyses were used to discriminate between the VOC profiles. The results indicated that the novel method described here could be used to detect Ganoderma disease and, more generally, for chemoecological studies of plant–pathogen interactions.     

A single dominant Ganoderma species is responsible for root rot of Acacia mangium and Eucalyptus in Sumatra

Ganoderma root rot is the most serious disease affecting commercially planted Acacia mangium in plantations in Indonesia. Numerous Ganoderma spp. have been recorded from diseased trees of this species and to a lesser extent Eucalyptus, causing confusion regarding the primary cause of the disease. In this study, a large collection of Ganoderma isolates were obtained from the roots of A. mangium showing early signs of root rot in disease centres in South Sumatra plantations. Isolates were also collected from Eucalyptus roots at Lake Toba in North Sumatra showing similar symptoms as well as from sporocarps connected to these samples. Phylogenetic analyses showed that a single Ganoderma sp., identified as G. philippii, is the major causal agent of Ganoderma root rot on A. mangium. Results from this study also showed that the isolates obtained for Eucalyptus trees in North Sumatra belong to G. philippii. Isolates from roots and connected fruiting bodies together with the morphology of the fruiting structures confirmed this identification. Symptoms associated with this pathogen are obvious and it should not be confused with other diseases. Other Ganoderma spp. found in disease centres are considered to be of minor importance and management strategies for root rot should be focused on G. philippii.     

Brenneria spp. and Rahnella victoriana associated with acute oak decline symptoms on oak and hornbeam in Iran

Decline diseases of forest trees are complex syndromes not attributable to single causal factors. In Iran, symptoms of decline disease have been observed in a number of native forest species including Quercus castaneifolia (chestnut‐leaved oak), Q. brantii (Persian oak) and Carpinus betulus (hornbeam). The symptoms are prevalent in the northern forests and the Zagros mountain forests. There are parallels between the disease in Iran and acute oak decline (AOD) reported in the UK, specifically the presence of weeping cankers, which have been attributed to a polybacterial complex wherein Brenneria goodwinii is considered a key necrogen. Based on the AOD symptomatology, and as a first step towards discovering potential causal agents of the stem weeping symptoms of affected trees in Iran, necrotic tissues were tested primarily for the presence of B. goodwinii. Symptomatic Q. castaneifolia and C. betulus from the Mazandaran Province and symptomatic Q. brantii from Kohgiluyeh and Boyerahmad Province were sampled. Isolation and culture on a selective medium yielded uniform bacterial colonies. Isolates were characterized using phenotypic and genotypic (DNA sequencing) tests. The isolates were phenotypically identical to members of Pectobacteriaceae and Yersiniaceae, specifically Brenneria and Rahnella spp. The nucleotide sequences of the 16S rRNA and housekeeping genes gyrB, infB and atpD (MLSA) amplified via PCR demonstrated that the isolates from the trees in Iran were indeed B. goodwinii, B. roseae subsp. roseae, Rahnella victoriana and an unknown species of Brenneria. Most bacteria isolated from non‐symptomatic trees were Gram‐positive, and Pseudomonas spp. were dominant, but bacterial species isolated from the diseased trees were not detected in healthy trees. Hypersensitivity response tests were positive, but inoculation on saplings was more variable with internal necrosis developing only once in the test period. Therefore, further testing is required. This is the first report of the incidence of B. goodwinii, B. roseae subsp. roseae, R. victoriana and Brenneria sp. associated with acute oak decline‐like symptoms on Q. castaneifolia, Q. brantii and C. betulus across the western forests of Iran and in the world.     

Identification and characterization of bacterial strains associated with diseased oak trees in Northern Iran

Oak decline syndrome is characterized by periodic occurrences of decline and death of oaks over widespread areas. An outbreak of a new emerging disease on oak trees was reported in the Hyrcanian forest of Iran (Mazandaran and Golestan provinces) that showed stem bleeding and canker symptoms. Bacterial isolates were characterized through biochemical and physiological tests, protein electrophoresis, DNA fingerprinting (rep‐PCR, ERIC and BOX primers) and sequencing of 16S rRNA and MLSA (multilocus sequencing analysis) for housekeeping genes (gyrB, infB and atpD). A complex community of the genus Brenneria spp. (Brenneria goodwinii, Brenneria roseae subsp. roseae, Brenneria sp. and Brenneria nigrifluens) and a few isolates in the genus Gibbsiella were identified as major groups involved. Isolate differentiation was more accurate using concatenated partial gene sequences within the main groups. All bacterial isolates showed hypersensitivity reactions (HR) on Pelargonium leaves (Pelargonium × hortorum). Pathogenicity studies of different Brenneria and Gibbsiella strains revealed that they have potential to cause the disease in oak seedlings and devastating oak canker and stem bleeding symptoms in northern Iran. Due to the presence of several potentially pathogenic agent(s) associated with the oak decline, identification of the principal agent(s) is of major interest. To our knowledge, this is the first report of potentially pathogenic bacteria associated with oak bleeding and canker in Iran.     

Outbreak of powdery mildew caused by Sawadaea negundinis on boxelder in Iran

Boxelder (Acer negundo) is planted as ornamental and shade tree in many countries including Iran. During July 2016, boxelder trees in parks and streets of different localities in East Azarbaijan province, Iran were found to be heavily infected with powdery mildew. Whitish powdery fungal mass developed mainly on the lower leaf surface. The infected young leaves became bubbled, curled and crinkled, giving affected plants an unpleasant appearance. During September, scattered, dark brown, spherical chasmothecia were produced on the lower leaf surface. The causal agent was identified as Sawadaea negundinis based on a combination of morphological characteristics of sexual and asexual morphs and sequence data of the rDNA internal transcribed spacer region (ITS1‐5.8S‐ITS2). This study provides the first report on the occurrence of S. negundinis on boxelder in Iran.     

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

The 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.     

Molecular and morphological characterization of Ektaphelenchoides maafiae n. sp. (Nematoda: Ektaphelenchinae) from northern forests of Iran

Ektaphelenchoides maafiae n. sp. was isolated during a survey of nematodes associated with bark samples of an oak tree (Quercus castaneifolia) in Gorgan, Golestan Province, northern Iran. The new species has a body length of 480–546 μm (in females) and 431–480 μm (in males). The cuticle is weakly annulated, with three lateral lines. Lip region offset. The stylet is 13–15 μm long without basal swellings. The excretory pore is located at the level of the metacorpus base to slightly posterior, and hemizonid is at 15–17 μm posterior to the excretory pore. The post‐uterine sac is short, 6–8 μm long. Spicules having rounded condylus, rostrum short, conical with bluntly pointed tip, a cucullus (apophysis) presented on the dorsal distal end. Male tail bearing four (2 + 2) caudal papillae, conical, with sharply pointed terminus. The new species is close to four known species of the genus, including E. hunti, E. ruehmi, E. caspiensis and E. poinari, but differs from them by body size, shape of tail terminus, stylet length, shape and size of spicules, length of post‐vulval uterine sac and number of caudal papillae. Phylogenetic analysis based on small subunit (SSU) and partial large subunit (LSU) sequences of rDNA confirmed its status as a new species.     

Management of fungal root‐rot pathogens in tropical Acacia mangium plantations

Acacia mangium is a fast‐growing tree species. It is mainly planted in large monocultures for pulpwood in South‐East Asia. Root rot has become the most economically damaging disease of this species with high tree mortality rates observed during second and third rotations. Two main types of root rots have been found in A. mangium, viz. brown root‐rot and red‐root disease caused by Phellinus spp. and Ganoderma spp., respectively. To assess the future management options for root rot of A. mangium, we review past and current disease‐management strategies for root rot in different temperate and tropical industrial tree crops. The efficacies of a wide range of silvicultural, chemical and biological options are detailed, and their potential utilization in managing root rot of A. mangium is discussed. We conclude that the current gaps in knowledge regarding identification, biology and disease epidemiology of the root‐rot pathogens will need to be addressed so that effective management options can be developed.     

Phylogenetic and pathogenicity evaluation of the marasmioid fungus Marasmius palmivorus causing fruit bunch rot disease of oil palm

Marasmius palmivorus is a marasmioid fungal species that exhibits parasitic behaviour, although most marasmioids are rarely parasitic. The fungus has been reported to cause fruit bunch rot disease of oil palm and coconut, but only a few studies on its pathogenic behaviour are available, particularly on oil palm. Hence, there is a need to assess the ability of the fungus to act as a pathogen and to study its molecular evolution and taxonomy. Nine isolates of M. palmivorus were successfully isolated from basidiocarps and diseased fruitlets of oil palm collected from oil palm plantations and were morphologically characterized on potato dextrose agar (PDA) followed by molecular identification based on nucleotide sequence alignments of internal transcribed spacer (ITS) regions of ribosomal RNA (rRNA) gene clusters with sequences from GenBank. Koch's Postulates confirmed that M. palmivorus could infect oil palm fruitlets with symptoms similar to those observed for bunch rot disease. Molecular phylogenetic studies using nucleotide sequences of ITS and the nuclear ribosomal large subunit (nLSU) showed that isolates sharing the same phenotypic characteristics of Marasmius palmivorus and Marasmiellus palmivorus are monophyletic and share a common ancestor. The fungus has also been shown to be more closely related to the genus Marasmius than Marasmiellus; therefore, we support retention of the taxon name of the pathogen causing bunch rot disease of oil palm as Marasmius palmivorus.     

“Candidatus Phytoplasma solani” associated with Eucalyptus witches’ broom in Iran

During summer of 2015, Eucalyptus camaldulensis plants showing witches’ broom, little leaf and general yellowing of the foliage were observed in west of Fars and Khozestan province of Iran. DNA from samples of 22 symptomatic and two asymptomatic trees was extracted and subjected to molecular analyses. Nested‐PCR test using R16F2n/R16R2 primers confirmed phytoplasma presence in 63% of symptomatic Eucalyptus plants. Sequence analysis along with virtual RFLP of the 16S ribosomal DNA allowed to classify three Eucalyptus witches’ broom strains into the “stolbur” (“Candidatus phytoplasma solani”) 16SrXII‐A subgroup. Comparison of the secA and secY gene sequences with sequences deposited in GenBank confirmed the phytoplasma identity. Real and virtual RFLPs of the amplified secY gene using HaeIII, MseI and RsaI restriction enzymes showed profiles indistinguishable from each other. This is the first study reporting E. camaldulensis as a new host species for “Ca. P. solani.”     

Occurrence and diversity of Phytophthora species in declining broadleaf forests in western Ukraine

In western Ukraine, forest decline and dieback of several broadleaved tree species have become increasingly evident during recent years, and surveys in some areas have shown symptoms indicative of Phytophthora infections. In this study, we aimed to determine the occurrence and diversity of Phytophthora species associated with several broadleaved tree species (Alnus glutinosa, Betula pendula, Castanea sativa, Fagus sylvatica and Quercus robur) from forest stands where dieback has been observed. Rhizosphere soil samples were collected from 14 forest stands during 2017 and 2018 and tested for the presence of Phytophthora species using morphological and molecular methods. Seven Phytophthora species (P. bilorbang, P. cactorum, P. gallica, P. gonapodyides, P. lacustris, P. plurivora and P. polonica), and two other clade six taxa were detected from the various forest types, several of which are probable agents responsible for decline. Four of the Phytophthora species (P. bilorbang, P. gallica, P. plurivora and P. polonica) have previously never been reported from broadleaf forests in Ukraine.     

Genetic diversity of pine‐parasitic nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus in China

The internal transcribed spacer (ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra‐ and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety‐one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS‐PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient (G_st) and gene flow (N_m) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, G_st and N_m of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short‐term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine‐parasitic nematode species, and will aid in controlling them in the future.     

An integrative study of Sakia sisanganensis n. sp. (Rhabditida: Tylenchidae) from Sisangan forest,Iran, and new morphological observations for the genus

Further data on the morphology (the lip region characters) and phylogeny of the genus Sakia are presented. The new observations were based on scanning electron microscopy (SEM) and light microscopy (LM). A new species, Sakia sisanganensis n. sp., was recovered from rotten wood of a dead beech tree (Fagus orientalis) in northern Iran, herein described and illustrated based on an integrative approach, that is morphological, morphometric and molecular characters. The new species is characterized by a combination of the following features: fine transverse striae and vestigial single band in the lateral field in SEM. Labial area dorso‐ventrally flattened. Oral region with two concentric hexagonal plates, the inner one apparently elevated. Amphidial openings short, slit‐like. Stylet delicate. Median bulb fusiform to spindle‐shaped with weak valvular apparatus. Spermatheca functional. Tail filiform with faintly pointed tip and males common. The new species was morphologically compared with four known species of the genus, viz., S. alii, S. arboris, S. castori and S. indica, all having indistinct lateral fields. Molecular phylogenetic analyses were performed based on the small subunit ribosomal RNA gene (SSU rDNA). In the Bayesian inference (BI), S. sisanganensis n. sp. with two isolates was strongly supported as a sister taxon of a clade harbouring S. arboris + Lelenchus species. However, in the maximum likelihood (ML) analysis, the new species formed a clade with S. arboris, thus supporting the reciprocal monophyly of the genera Sakia and Lelenchus. Accordingly, the test of monophyly was performed (using Bayes factor) and the results did not reject the monophyly of sakia (i.e., S. sisanganensis n. sp. and S. arboris as sister taxa) based on the currently available data.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

Agrobacterium pusense,a new plant tumour‐inducing pathogen isolated from Lawson cypress

Lawson cypress (Chamaecyparis lawsoniana), an important landscape tree, is widely planted in gardens and parks throughout Iran. Crown gall disease on Lawson cypress trees was observed in Sari and Juybar Counties, Mazandaran province, northern Iran, in 2017. Isolation from galls on potato dextrose agar (PDA) containing CaCO₃ yielded bacterial colonies, the predominant types of which were purified and selected for characterization. The isolates were Gram‐negative, oxidase positive, able to grow in 2% NaCl and produced 3‐ketolactose. They hydrolysed esculin, casein and arbutin but not starch, gelatin or Tween 80. Two representative isolates were selected for PCR amplification and sequencing of DNA gyrase subunit B (gyrB) gene. In the phylogenetic tree based on the partial sequence of the gyrB gene, isolates KH1 and KH2 clustered with Agrobacterium pusense. The pathogenicity of all isolates was confirmed by inoculation on Jimsonweed (Datura stramonium) and carrot discs (Daucus carota). Confirmation of the presence of genes involved in pathogenicity was made by performing PCR with the virD2A/virD2C and VCF/VCR primer pairs which resulted in amplification of the expected 224 and 730 bp fragments in all studied isolates, respectively. A. pusense was therefore identified as the causal agent of crown and stem gall of Lawson cypress. This appears to be the first report on the natural occurrence of crown gall disease on Lawson cypress and the first record of a plant disease caused by A. pusense.     

Role of site in the mortality and production of Acacia mangium plantations in Indonesia

In Indonesia, Acacia mangium plantations exceed 1.6 Mha contributing approximately 3.5% of the country’s GDP. The viability of these plantations is increasingly threatened by fungal pathogens, insect pests, squirrels, monkeys, elephants and wind damage. Studies indicate that the problem is growing and in some areas, fungal pathogens such as Ganoderma and Ceratocystis species have contributed up to 50% tree mortality by the fourth rotation. Multiple statistical procedures were employed to examine the influence of soil and topographical properties on tree survival (trees ha^?1), wood production (m³ ha^?1), and mortality associated with Ganoderma root rot, Ceratocystis wilt and by wind. Soil family level was found to be a good indicator of tree mortality. Plots with fine-loamy Typic Kandiudult soils had the highest tree survival and mortality associated with species of Ganoderma and Ceratocystis, but had the lowest incidence of mortality by wind. The degree of association between soil and topographic variables with tree survival, wood production and the cause of mortality were poor and inconsistent. Tree survival was slightly higher on upslope areas away from valley bottoms, and drier mid-slopes, ridges and hilltops, and very low pH (<3.3) soils. Wood production was also slightly higher in drier, elevated locations, away from valley bottoms. Mortality by wind was slightly higher in moist, poorly drained, low-lying valley bottoms and topographically flat areas. Our ability to further pinpoint the influence of topography and soil attributes on wood production and cause of mortality was greatly compromised by the lack of site-specific soil data, and potential misclassification of the cause of mortality. This study could not reliably or consistently relate tree survival, wood production or the cause of mortality to any one, or combination of, soil and topographic variables.     

New hosts for Lecanosticta acicola and Dothistroma septosporum in newly established arboreta in Spain

A historical outbreak of needle blight disease was recorded during 2018 to 2019 in plantations of Pinus radiata and Pinus nigra in the North of Spain. The main pathogens involved in this historical outbreak were identified as Lecanosticta acicola and Dothistroma septosporum. Recently, a variety of tree species in three arboreta planted between 2011 and 2013 in the Basque Country as part of the European project REINFFORCE were showing symptoms of needle blight and defoliation. The aim of this study was to determine which pine species were affected with these pathogens. Tree species sampled included several provenances of P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda. Using molecular identification methods, Lecanosticta acicola was confirmed infecting Pinus brutia (Provenance: Alexandropolis, Greece and var. eldarica, Crimea) and represents a new host species for this pathogen. Pinus elliottii (Provenance: Georgia, USA) and P. ponderosa (Provenance: Central California, USA) are new host reports of L. acicola for Spain. Dothistroma septosporum was found for the first time on P. brutia (Provenance: Marmaris, Turkey) and P. ponderosa (Provenance: Oregon, USA) in Spain and was also detected infecting P. nigra (Provenance: Sologne Vayrières, France).     

Winter Browsing by Large Herbivores on Planted Deciduous Seedlings in Southern Sweden

Deciduous seedlings are valuable for the forest industry and, at the same time, often palatable for large herbivores. Therefore, browsing on seedlings of seven deciduous tree species and on Picea abies(L.) Karst., as a reference species, was tested in a field experiment with the species presented at feeding stations. Twenty stations, each with one seedling per species, were established in each of 15 clear-cuts in southern Sweden. The planting took place in October. Depending on species, between 16 and 85% of the seedlings were browsed after one winter. The descending order, based on number of browsed seedlings, was Quercus robur L . > Alnus glutinosa (L.) Gaertn.> Fagus silvatica L.> Tilia cordata Mill.> Prunus avium L.> Betula pendula Roth> Picea abies (L.) Karst. > Fraxinus excelsior L. On 83% (range 45-100%) of the browsed seedlings the leader was damaged. On average, browsing reduced seedling hight by 32% (range 12-47%). Within species, initially taller seedlings were more often selected than shorter ones.     

Parasitoid complex (Hymenoptera, Braconidae, Aphidiinae) of Aphis craccivora Koch (Hemiptera: Aphidoidea) in Iran

The parasitoids (Hymenoptera, Braconidae, Aphidiinae) of the cowpea aphid, Aphis craccivora Koch, were investigated throughout two far distant provinces, Tehran and Sistan-Baluchestan. Colonies of the cowpea aphid were collected from different host plants and reared under laboratory conditions until the parasitoids emerged. The aphids were parasitized by an expected range of parasitoids. The species were Aphidius colemani Viereck, Lysiphlebus fabarum (Marshall), Lysiphlebus confusus Tremblay and Eady, Lysiphlebus testaceipes (Cresson), Binodoxys acalephae (Marshall), Binodoxys angelicae (Haliday), Praon volucre (Haliday) and Ephedrus persicae Froggatt. L. testaceipes is reported here for the first time in Iran. The most common species was L. fabarum followed by B. acalephae and L. confusus. A key is provided for identification and host plant associations are considered.