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.     

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.     

Characterization of endophytic bacteria with plant growth promotion and biological control potential isolated from walnut trees

In this study, a total of 68 endophytic bacteria were isolated from different tissues of walnut trees. About 55% and 22% of the strains had the ability to produce indole acetic acid and gibberellic acid, respectively. The capability of isolates to solubilize phosphate, growth on N‐free medium, siderophore, protease and lipase production was varied. Based on phenotypic grouping and plant growth promotion properties, twelve isolates were selected and 16S rRNA gene‐based phylogenetic analysis revealed that strains showed 99%–100% similarity to Pseudomonas, Bacillus, Arthrobacter, Roseomonas and Streptomyces genera. Amongst the selected strains, PS12, KS54, JS66 and KS74 showed root and shoot growth enhancement of poplar cutting. NS70, KS54 and PL36 strains showed antagonistic activity against Pseudomonas syringae pv. syringae;RR47, KS74 and NR69 strains had inhibition effects against Brenneria nigrifluens; and JS66 and RR26 strains had antagonistic activity against both phytopathogens under in vitro conditions. This is the first reported study to elucidate the endophytic bacterial diversity associated with walnut trees with beneficial attributes.     

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.     

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.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

The first record of Botryodiplodia canker in Poland

This study describes the first observation of Botryodiplodia canker in the Western Carpathians in south‐eastern Poland caused by Botryodiplodia hypodermia (Sacc.) Petr. (syn. Sphaeropsis hypodermia, S. ulmicola). The canker occurred on an approximately 17‐year‐old Ulmus glabra sapling in a mixed conifer/deciduous stand with elm trees severely damaged by Dutch elm disease. This paper describes disease symptoms and provides information on the macro‐ and micromorphology of the fungus isolated from the cankered tissues. The results of BLAST search using DNA sequences obtained for our cultures and subsequent phylogenetic positioning of the fungus among closely related Botryosphaeriaceae indicate that the species is much more closely related to Phaeobotryon than to the other Botryodiplodia or Sphaeropsis species. Moreover, a total of 16 polymorphisms within the ITS region were detected between S. ulmicola associated with Botryodiplodia canker in North America and B. hypodermia associated with the canker observed in Poland. Thus, the “European” variant of “Sphaeropsis” ulmicola can now be easily identified with our barcode sequences. The Botryodiplodia canker is much less prevalent in Europe than in North America. Differences in virulence of “American” and “European” linages and differences in susceptibility of various elm species may be the reason for the higher prevalence of the disease in North America.     

An agglutination test for fast and specific detection of Erwinia psidii

Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 10⁵ colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.     

First detection of Neoscytalidium dimidiatum associated with canker disease in Egyptian Ficus trees

In September 2013, a canker disease of Ficus nitida and F. benjamina was reported for the first time in Assiut governorate, Egypt. Infected samples were collected from various locations. Pure cultures of a fungus were isolated on potato dextrose agar at 25°C from diseased plants. Morphological investigation and DNA sequencing showed that the causal agent was Neoscytalidium dimidiatum. A pathogenicity test conducted using 2‐year‐old plants of each host gave 70%–80% infection, and the pathogen was reisolated from the inoculated plants. A pure culture of N. dimidiatum was deposited in the culture collection of the Assiut University Mycological Centre (AUMC) under the code AUMC 9293 and the ITS sequence was placed in NCBI under accession number KX985929 .     

Isolation and identification of endophytic bacteria with plant growth promoting and biocontrol potential from oak trees

Samples of roots, leaves and stems of healthy oak trees were collected from various locations in the Baneh and Marivan regions, Iran. In total, 63 endophytic bacteria were isolated and grouped according to phenotypic properties. Seven selected isolates were further identified by partial sequencing of the 16S rRNA gene. Isolates Pp54, Pp88, Pp95 and Pp177 were Pseudomonas spp., isolates Sm59 and Sm79 were Stenotrophomonas spp., and isolate Bf 172 was a Bacillus sp. The ability of these isolates for plant hormone production such as auxin and gibberellin was evaluated, along with siderophore production, phosphate solubilization, atmospheric nitrogen fixation, protease and hydrogen cyanide production. All strains produced auxin and gibberellin in different amounts. Atmospheric nitrogen fixation ability was positive for strains Sm79, Pp54, Pp88 and Pp95. All strains except Sm79 solubilized phosphate. Strains Sm59, SM79, Pp88, Pp177 and Bf172 produced protease. Pp88, Pp95, Bf172 and Pp177 were able to produce siderophore. Strains Sm79 and Pp95 released low concentrations of hydrogen cyanide. Amongst the strains tested, Pp95, Pp88, Pp177 and Sm79 had different inhibitory effects on the bacterial plant pathogen, Pseudomonas syringae pv. syringae under in vitro conditions. This is the first reported case of endophytic Bacillus, Pseudomonas and Stenotrophomonas strains from oak trees in Iran.     

Evaluation of fungicides for the control of Botryosphaeria corticola on cork oak in Catalonia (NE Spain)

Botryosphaeria canker of cork oak (Quercus suber L.), caused by Botryosphaeria corticola, is a well-known factor contributing to the decline of this forest species and is a serious disease in the main cork-producing countries of the Western Mediterranean basin. In this work, fungicides with the potential for controlling the disease were selected using a combination of in vitro tests and field trials. Fourteen fungicides in different chemical families were tested in vitro to evaluate their effect on inhibition of mycelial growth. The most effective fungicides were benomyl, carbendazim, cyprodinil + fludioxonil, thiabendazol and thiophanate-methyl; these compounds were then further tested under field conditions. In the field trials, carbendazim and thiophanate-methyl were the most effective fungicides, as shown by efficacy indices, in reducing debarked trunk surface affected by cankers (76% and 50–65%, respectively). These results suggest that the occurrence of Botryosphaeria canker on cork oak can be reduced effectively with the application of selected fungicides after cork removal from the trees.     

Investigation of the phenotypic and genetic properties of Brenneria nigrifluens strains,the causal agent of walnut bark canker in Kurdistan province,Iran

Phenotypic and genetic properties of Brenneria nigrifluens, the causal agent of walnut bark canker disease were studied using a combination of key biochemical tests, analysis of whole‐cell protein extracts and genetic fingerprints based on DNA primers corresponding to conserved motifs in bacterial repetitive (repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus and BOX) elements and to the 16s rRNA gene. Phenotypic properties indicated that 11 strains showed a high degree of similarity and <5% of isolates showed different properties such as utilization of dulcitol and inositol. A majority of isolates produced rep‐PCR profiles similar (>80%) to a B. nigrifluens reference strain. This study showed that the majority of strains had 0–3 plasmids with estimated sizes ranging 9–32 Kb. Strains were clustered into two and three groups by whole‐cell protein analysis and rep‐PCR, respectively.     

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.     

The true nature of Ganoderma in Iran: Taxonomy based on ITS and mtSSU rDNA

The genus Ganoderma Karst. has broad‐spectrum usage in biotechnology, medicine and the food industry. The complexity of the morphology within species has led to uncertain identification in the past, but recent advancements in molecular identification methods have provided scientists with the opportunity to better understand the taxonomy of the species. The present study attempts for the first time to elucidate the distinctiveness of the Ganoderma species growing in Iran concerning those elsewhere in the world based on mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and internal transcribed spacer (ITS) sequence data. The results disclosed that the G. lucidum Karst. samples collected in Iran are more similar to the European Ganoderma species than to the Asian (Chinese) one used in this study.     

Relative susceptibility of oaks to seven species of Phytophthora isolated from oak forest soils

Isolates of Phytophthora cambivora, P. cinnamomi, P. citricola, P. europaea, P. quercetorum and two unidentified species were tested for their pathogenicity to eastern US oak species by root and stem inoculations. Experiments were conducted during two different periods and included 1‐, 2‐ and 20‐year‐old oaks grown under greenhouse and field conditions. Species of Phytophthora were pathogenic in varying degrees to the oak species tested. All species were pathogenic to fine and taproots of at least one oak species. The fine root damage caused by the species of Phytophthora ranged from 9 to 55% when compared to the controls. Roots were more susceptible during the fall inoculation period than the summer. With exception of Phytophthora sp1 and P. quercina‐like, all species of Phytophthora were pathogenic to oak stems with P. cinnamomi and P. citricola being the most aggressive. Quercus montana and Q. rubra were the most susceptible oak species to stem inoculation. Lesion sizes were considerably larger when 20‐year‐old trees were inoculated. Generally, no significant differences in lesion sizes were detected in greenhouse tests when the summer and fall inoculation periods were compared. However, on 2‐year‐old field‐grown seedlings, lesion sizes were considerably smaller or not significantly different from controls during the fall inoculation period, suggesting lower, late season temperatures may restrict lesion development.     

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.     

Providencia rettgeri as the causal agent of the brown slime flux of Populus tomentosa

Brown slime flux seeps slowly out of wounds and flows down the bark of roadside Populus tomentosa in Beijing, China. Two bacterial isolates, SL2‐2 and SL3‐3, obtained from the brown slime flux were identified as Providencia rettgeri based on 16S rRNA gene sequencing. After inoculation into the bark of roadside P. tomentosa with sterile deionized water as a control, both SL2‐2 and SL3‐3 triggered the seepage of brown slime flux from the wounds; no slime flux arose from the control. In conclusion, P. rettgeri was the pathogenic bacterium causing brown slime flux on P. tomentosa probably by inhibiting wound healing. To our knowledge, this is the first report of P. rettgeri as a pathogen of plants.     

Differences in composition of endophytic mycobiota in twigs and leaves of healthy and declining Quercus species in Italy

The composition of the endophytic fungal assemblages in Quercus cerris, Q. pubescens and Q. robur, three oak species most susceptible to decline, was investigated in Fagarè (Padova), Ulignano (Pisa), and Radda in Chianti (Firenze). The endophytic assemblages were studied as a function of health status of the whole tree and of twigs and leaves. The effect of station altitude was tested on Q. cerris. Samples were gathered from healthy and declining trees. In April (bud opening time) 20 twigs and 10 leaves were collected from each tree. Fungi were isolated from twig segments and leaf portions. A total of 23 fungal genera with 27 different species were isolated, in addition to a few sterile mycelia. Eleven species were found to be common to all three oak species, while other species were found only on one oak species, suggesting host specificity. Among the fungal species isolated, some were known to be pathogenic (Apiognomonia quercina, Colpoma quercinum, Diplodia mutila, Phomopsis quercina). The colonization frequency (CF) of pathogenic species varied between 0.9% for A. quercina in Q. cerris and 60.2% for P. quercina in Q. robur. Analysis of variance showed a statistically significant difference among the oak species tested. The CF was higher in declining trees in comparison with healthy trees, and also in twigs vs. leaves. The CF was found to be higher for Q. cerris trees growing at sea level as compared with those situated at an elevation of 350–400 m. In stands where, on account of particular ecological conditions, pathogenic behaviour is displayed simultaneously by more than one endophytic fungal species, trees accentuate their decline.     

Diplodia corticola and Phytophthora cinnamomi: the main pathogens involved in holm oak decline on Caprera Island (Italy)

Since 2008, severe and widespread tree decline and mortality has been observed at the main growing Quercus ilex L. (holm oak) forest on Caprera Island, Italy. To clarify the symptomatology and aetiology of this phenomenon, field surveys and isolations from symptomatic trees were carried out in summer 2010. Affected trees exhibited crown thinning, branch dieback, sunken cankers, epicormic shoots, exudates on branches and trunk, root losses and sudden death symptoms. Four fungal species belonging to Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, D. seriata and Neofusicoccum parvum, were isolated from cankers on trunk and branches, whereas three species of Phytophthora, namely P. cinnamomi, P. cryptogea and P. gonapodyides, were isolated from fine roots and rhizosphere soil samples. Isolates were identified using both morphological analysis and DNA‐based techniques. Pathogenicity trials on holm oak seedlings showed that all the isolated species are pathogenic. D. corticola proved to be the most aggressive species. Our results provide the first evidence for a combined involvement of D. corticola and P. cinnamomi in the aetiology of holm oak decline in Italy and suggest that these pathogens are not only important contributing factors in the onset of long‐term tree decline, but also may cause the rapid devastation of extensive oak ecosystems.