Rapid detection and identification of ‘Candidatus Phytoplasma pini’‐related strains based on genomic markers present in 16S rRNA and tuf genes

In order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini ’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma .     

Molecular identification of a phytoplasma associated with Elm witches’‐broom in China

Elm samples with and without witches’‐broom symptoms (EWB) were collected from Tai’an and Zhaoyuan, Shandong Province, China. Phytoplasmal cells were observed in the phloem cells of symptomatic plants under electron microscope. Specific fragments of about 1.2 kb in length were amplified with nested‐PCR from symptomatic samples, while no fragment was obtained from healthy plants. The 16S rRNA gene sequences of the phytoplasmas associated with elm witches’‐broom in Tai’an (EWB‐TA) and Zhaoyuan (EWB‐ZHY) had high similarities, and formed a sublineage in phylogenetic tree, with members of subgroup B or D of aster yellows group (16SrI). Computer simulated restriction fragment length polymorphism analysis of 16S rRNA gene revealed that EWB‐TA and EWB‐ZHY patterns had similarity coefficients of 1.00 with the pattern from the representative strains of subgroup 16SrI‐B, and had a similarity coefficient of lower than 0.97 with representatives of other subgroups. These results indicated that the phytoplasma strain associated with elm witches’‐broom in China was very closely related to ‘Candidatus Phytoplasma asteris’ OAY, belonging to subgroup‐B of aster yellows group (16SrI‐B). This is the first report of a phytoplasma associated with elm witches’‐broom disease in China.     

“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.”     

First report of 16Sr II‐C subgroup phytoplasma association with Acacia mangium in Tripura,India

Leaf yellowing symptoms were observed on Acacia mangium in the Sipahijala district of Tripura, India, during June 2017. Symptomatic and asymptomatic leaf samples (three of each) were collected from roadside trees of A. mangium for DNA extraction using the CTAB method. Amplicons of ~1.25 kb and ~480 bp were detected in all the symptomatic samples using the phytoplasma‐specific universal 16S rRNA and secA gene primers. Pair wise sequence analysis of 16S rRNA gene sequences, virtual RFLP and phylogenetic analysis revealed that the phytoplasma strain associated with A. mangium belonged to phytoplasma subgroup 16SrII‐C. This is the first report of an association between the 16SrII‐C subgroup and A. mangium leaf yellowing.     

A new phytoplasma associated with witches’‐broom on Japanese maple in China

In September 2011, five Japanese maple (Acer palmatum Thunb.) trees with symptoms of witches’‐broom were observed growing near each other at a maple grove in Northwest A&F University, Yangling, Shaanxi Province, China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants under transmission electron microscope (TEM). The presence of phytoplasma was further confirmed by a nested polymerase chain reaction (PCR), which amplified a 1.2‐kb fragment using universal primer pair R16mF2/R16mR1 followed by further amplification using primer pair R16F2n/R16R2. Phylogenetic analysis and gel‐based restriction fragment length polymorphism (RFLP) analysis demonstrated that the Japanese maple witches’‐broom was associated with phytoplasma belonging to subgroup 16SrI‐D. This is the first report of a phytoplasma disease of Japanese maple.     

Identification of ‘Candidatus phytoplasma ziziphi’ associated with persimmon (Diospyros kaki Thunb.) fasciation in China

Persimmon (Diospyros kaki Thunb.) trees, with fasciation symptoms (PF), were observed in an orchard located in suburban Tai'an, Shandong Province, China. A specific fragment of the phytoplasma 16S rRNA gene, approximately 1.2 kb in length, was amplified from two symptomatic plants via nested polymerase chain reaction, while no fragment was obtained from healthy controls. The two samples (PF1 and PF2) resulted with 99.5% nucleotide sequence identity. Phylogenetic and restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences revealed that PF1 was a member of ribosomal subgroup B of the elm yellows group (16SrV), and PF2 may represent a novel subgroup within the 16SrV group, designed as 16SrV‐I. This is the first report of phytoplasmas of the 16SrV group associated with persimmon fasciation disease.     

Molecular characterization of a phytoplasma from the aster yellows (16SrI) group naturally infecting Populus nigra L. ‘Italica’ trees in Croatia

Leaf and branch samples were collected from 10 Populus nigra L. ‘Italica’ trees found in the Zagreb urban area. One of the P. nigra L. ‘Italica’ trees exhibited leaf yellowing, overall sparse foliage, stunting and decline. Two methods for the nucleic acid extraction in the phytoplasma detection from P. nigra were compared. A phytoplasma from the aster yellows group (16SrI) was detected by PCR in the symptomatic as well as in four apparently asymptomatic plants. The pathogens are classified, by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene plus the spacer region, as members of a newly described subgroup 16SrI‐P. Phylogenetic analysis of 16S ribosomal and spacer region sequence confirmed their close relationship with the other members of the aster yellows group. However, RFLP analyses of other conserved genes such as tuf, BB88 and ribosomal protein rpL22 gene, clearly confirmed that this is a molecularly distinguishable phytoplasma belonging to a new ribosomal protein subgroup designated rp‐O.     

Salix tetradenia Hand.‐Mazz: a new natural plant host of ‘Candidatus phytoplasma’

This article reports Salix tetradenia Hand.‐Mazz as a new host of Candidatus phytoplasma and demonstrates its association with witches' broom disease on S. tetradenia plants. Plants exhibited typical visual symptoms of phytoplasma with virescence, abnormality of flowers and witches' broom, and phytoplasma bodies were observed by transmission electron microscopy. Products of 1.2 kb were amplified by nested PCR using phytoplasma universal primer pairs R16F2n/R16R2, but no amplification products were obtained from symptomless plants. The sequence analysis of three 16S rDNA isolates showed 99.84%, 99.68% and 99.76% identify, respectively, with the homologous gene (nc_005303) of member of ‘Candidatus phytoplasma asteris’ (16SrI) group. Phylogenetic and virtual computer‐simulated restriction fragment length polymorphism analysis of the 16S rRNA, tuf and rp gene sequences confirmed that this phytoplasma clustered in the 16SrI‐B subgroup. These results indicated that the diseased S. tetradenia plants were infected by a phytoplasma of the 16SrI group. This is the first report on the occurrence of phytoplasma disease on S. tetradenia worldwide.     

Fusarium species in declining wild apple forests on the northern slope of the Tian Shan Mountains in north‐western China

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.     

Molecular identification and characterization of a new phytoplasma strain associated with Chinese chestnut yellow crinkle disease in China

Chinese chestnut trees (Castanea mollissima BL.) planted in a suburb of Beijing, China developed symptoms including yellowing, leaf crinkling, little leaf, shortened internodes, and empty burrs. Transmission electron microscopy revealed presence of phytoplasma cells in phloem sieve elements of the symptomatic chestnut trees. Molecular cloning and sequence analysis of PCR‐amplified near‐full length 16S rRNA gene indicated that the phytoplasma associated with the Chinese chestnut yellow crinkle disease is closely related to Japanese chestnut witches’‐broom phytoplasma. This is the first report of a phytoplasmal disease in Chinese chestnut trees.     

Genetic diversity of apple‐ and crabapple‐infecting isolates of Venturia inaequalis in Pennsylvania,the United States,determined by microsatellite markers

Apple scab caused by Venturia inaequalis is the most destructive disease of apple worldwide. In this study, genetic diversity of 101 V. inaequalis isolates from cultivated apples and ornamental crabapples in Pennsylvania (PA, USA) was characterized using 14 microsatellite markers. A total of 157 alleles ranging from 5 (Vitg9/99) to 26 (Vica10/154) per locus were detected. Regardless of the host of origin, isolates were grouped into five clusters, which were largely supported by STRUCTURE and principal coordinate analysis. Cluster analyses based on genetic distances and population structure analysis suggest very small differentiation (PhiPT ranging from 0.016 to 0.103, depending on the population comparison) between apple and crabapple isolates of V. inaequalis. Pairwise comparisons among populations from different locations showed very low differentiation, and POPGENE analysis indicated frequent migration of alleles (Nm = 1.47). In pathogenicity tests using a detached leaf assay, isolates of V. inaequalis from crabapple caused characteristic scab symptoms on apple and were highly virulent. Results of the study indicate that scab lesions in crabapple trees in close vicinity to apple orchards could serve as reservoirs for spread of the pathogen. Movement of inoculum among locations and between hosts may be responsible for the limited population structure observed. Understanding the population structure of V. inaequalis isolates is significant for apple scab management as crabapples are often used as pollinizers and rootstock in apple orchards, and as ornamental trees.     

Molecular characterization of a phytoplasma associated with Euonymus bungeanus witches’ broom in China

Euonymus bungeanus plants exhibiting symptoms of abnormal branches, small leaves and phyllody, which is indicative of E. bungeanus witches’ broom (EbWB) disease, have recently been found in Beijing, China. A phytoplasma from symptomatic E. bungeanus plants was identified by 16S rRNA polymerase chain reaction (PCR) using the phytoplasma‐specific universal primer pair R16mF2/R16mR1. Inoculation of healthy E. bungeanus plants by grafting with diseased scions was also performed. The rp and secY genes of the EbWB phytoplasma were cloned and sequenced as was the 16S rRNA gene. Sequence and phylogenetic analyses of 16S rRNA, rp and secY genes indicated that the phytoplasma associated with E. bungeanus belongs to the 16SrV‐B, rpV‐C and secY‐C subgroup, the same subgroup as the jujube witches’ broom (JWB) phytoplasma that is widely distributed among jujube trees in China. Comparative analyses based on virtual restriction fragment length polymorphism (RFLP) showed that the EbWB phytoplasma is more closely related to another 16SrV‐B subgroup strain: RPWB (Robinia pseudoacacia witches’ broom). To the best of our knowledge, this is the first report of a witches’ broom phytoplasma in E. bungeanus in China, and the findings add a new cultivated plant species to the already broad natural host range of 16SrV‐B subgroup phytoplasmas.     

Apple stem grooving virus naturally infects Himalayan wild cherry (Prunuscerasoides D. Don)

Himalayan wild cherry (Prunus cerasoides), widely distributed in the Himalayas, was found to exhibit severe virus‐like symptoms (chlorotic spots, chlorosis along the margins of the leaf and necrotic spots). Of 47 symptomatic and asymptomatic leaf samples tested through DAS‐ELISA, dot‐blot hybridization and RT‐PCR, only three were found to be positive for Apple stem grooving virus (ASGV) infection. The complete coat protein gene from all the three positive samples was molecularly characterized and sequencing of the amplicons confirmed presence of the virus. The three characterized isolates of Himalayan wild cherry (HWC‐15, HWC‐16 and HWC‐47) grouped with the ASGV apple isolates from India, Brazil and China. Of the three, two isolates (HWC‐15 and HWC‐47) shared around 100% sequence identity among themselves while 96.2% with the third isolate (HWC‐16) (both at nucleotide and amino acid level), respectively. While they all shared an overall identity of around 92.8–99% at (aa) and 86.5–99.5% at (nt) with rest of the isolates from different hosts and geographical locations. Experimental host range of the variant HWC‐16 isolate identified C. amaranticolor, C. sativus, C. quinoa, P. vulgaris, N. benthamiana and N. glutinosa as positives for the ASGV isolate‐inducing epinasty, symptomless carrier, chlorotic spots, interveinal chlorosis, chlorotic spots and chlorotic patch. To the best of our knowledge, this is a first report of natural infection of ASGV on Himalayan wild cherry.     

High genetic variation in a small population of Cherry leaf roll virus in Betula sp. of montane origin in Corsica

Cherry leaf roll virus (CLRV) infection is common in Betula pendula and B. pubescens in Middle and North Europe, easily observable by chlorotic leaf vein banding, mottling and leaf roll, partially adherent with progressive loss of vitality or death of twigs and branches. In Fennoscandia, a severe viral epidemic in various birch species in forest stands, public greens and roadsides is associated with CLRV. In Corsica, CLRV‐typical symptoms were observed on birch trees (Betula sp.) in a montane stand (1470 m) at Col de Vergio. CLRV was detected by RT‐nested PCR in all leaf samples from 11 randomly selected birch trees exhibiting characteristic symptoms. Along with the fact that this is the first report of CLRV in Betula sp. of both montane and Mediterranean origins, remarkably high genetic variation and a new distinct phylogenetic cluster are comprised by a small randomly sampled CLRV population that has evolved in one of the few scattered birch stands in Corsica.     

Detection of phytoplasma infections in declining Populus nigra ‘Italica’ trees and molecular differentiation of the aster yellows phytoplasmas identified in various Populus species

A disease of Populus nigra‘Italica’ associated with foliar yellowing, sparse foliage, stunting, dieback, and decline was observed in south-western Germany; a witches’ broom disease of Populus alba that is known in other countries was also detected in Hungary and Germany. The aetiology of the diseases was studied by fluorescence microscopy and polymerase chain reaction (PCR) amplification. Using fluorescence microscopy, phytoplasmas could be detected only in P. alba. However, most diseased trees of P. nigra‘Italica’ tested phytoplasma-positive by PCR. In some of the trees the phytoplasma numbers were so low that nested PCR was required to detect the infection. Very low phytoplasma numbers were also observed in diseased Populus tremula. The identity of phytoplasmas from P. nigra‘Italica’ sampled in Germany and France, P. alba and also P. tremula was examined by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA. In all poplars, phytoplasmas of the aster yellows group were detected. However, three different RFLP groups were identified that consisted of (1) French strains from P. nigra‘Italica’, (2) German strains from P. nigra‘Italica’ and (3) strains from P. alba and P. tremula. The profile observed in the last group was probably the result of sequence heterogeneity in the two 16S RNA genes.     

Natural infection of Fraxinus excelsior seeds by Chalara fraxinea

The occurrence of Chalara fraxinea, the fungus responsible for dieback of European ash (Fraxinus excelsior), was investigated in the current and previous seed years collected from symptomatic trees in Latvia and Sweden using molecular techniques (DNA extraction, ITS‐PCR, Sanger sequencing). Molecular analysis of seeds revealed the presence of 30 different fungal taxa. Chalara fraxinea was detected in 8.3% of seeds tested from the current year originating from Latvia. The presence of C. fraxinea in seeds of F. excelsior is of great concern to phytosanitary protection authorities in countries outside the current zone of infestation.     

Phytophthora pseudosyringae associated with the mortality of Nothofagus obliqua in a pure stand in central‐southern Chile

Mortality of Nothofagus trees in the southern‐central Chile region has been observed for over 30 years. A field survey conducted in 2013 detected partial defoliation and bleeding cankers on Nothofagus obliqua in a pure stand in the Nahuelbuta coastal ranges of the Biobío region. A Phytophthora sp. was isolated from stem cankers and soil samples around symptomatic N. obliqua trees: All isolates were identified as Phytophthora pseudosyringae. These isolates were pathogenic on 1‐year‐old N. obliqua and Nothofagus alpina, and on detached twigs of adult N. obliqua and Nothofagus dombeyi trees. This paper is the first to report association and pathogenicity of P. pseudosyringae with N. obliqua, N. alpina and N. dombeyi native to the Biobío region of Chile. The potential of P. pseudosyringae to cause damage in natural Nothofagus stands in Chile must be determined.     

Genetic variability of Alder yellows phytoplasma in Alnus glutinosa in its natural Spreewald habitat

The genus ‘Candidatus Phytoplasma’ comprises wall‐less bacteria colonizing the phloem of plants and insect tissues. Phytoplasmas are associated with diseases in over 1000 plant species worldwide, including many important crops as well as forest trees. Alder yellows (AldY) phytoplasma, which frequently infects Alnus spp., is closely related to the economically important phytoplasma causing Flavescence dorée (FD) in grapevines. In a natural habitat (Spreewald, Brandenburg, Germany), 57 Alnus glutinosa (black alder) trees were examined for phytoplasma infection in summer 2013. No phytoplasma typical infection‐associated symptoms such as yellowing and decline were observable in this natural swamp‐alder area. Amplification followed by a restriction fragment length polymorphism, and a sequence analysis of the 16S rDNA, allowed for the detection of AldY phytoplasmas in all examined trees and their assignment to the taxonomic group 16SrV‐C. Additional analyses of the non‐ribosomal marker gene methionine aminopeptidase (map) revealed diverse strains as well as mixed infections with closely related AldY strains, and the strains were assigned to phylogenetic clusters closely related to German Palatinate grapevine yellows, AldY or FD strains. The results confirmed that AldY phytoplasmas infection in A. glutinosa is prevalent. The results also indicate a presence of an established phytoplasma population in chronically infected black alder.     

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.     

Previously unrecorded low‐temperature Phytophthora species associated with Quercus decline in a Mediterranean forest in eastern Spain

Oak decline has been a serious problem in Europe since the beginning of the twentieth century. In south‐west Spain, Quercus ilex and Q. suber are the main affected species, and their decline has been associated with Phytophthora cinnamomi. During the last 10 years, a severe decline of Q. ilex and Q. faginea accompanied by a significant decrease in the production of acorns affecting natural regeneration was observed in the eastern part of the Iberian Peninsula. Therefore, the aim of this study was to investigate the possible involvement of Phytophthora spp. in the decline. A forest in the Natural Park ‘Carrascar de la Font Roja’ in Comunidad Valenciana (eastern Spain), which is dominated by Q. ilex and Q. faginea, was surveyed during 2010–2011. Symptomatic trees showed thinning and dieback of the crown, withering of leaves and death. An extensive loss of both lateral small woody roots and fine roots and callusing or open cankers on suberized roots were observed. Soil samples containing fine roots were baited using both Q. robur leaves and apple fruits. Six Phytophthora species were isolated: P. cryptogea, P. gonapodyides, P. megasperma, P. quercina, P. psychrophila and P. syringae. These are the first records of P. quercina and P. psychrophila on Q. faginea, of P. quercina in Spain and of P. psychrophila in mainland Spain. A soil infestation trial was conducted for 6 months under controlled conditions with 1‐year‐old seedlings of Q. ilex and Q. faginea. Phytophthora cinnamomi was included in the pathogenicity test for comparison. The results showed that Q. ilex seedlings were generally more susceptible to infection than Q. faginea with P. cinnamomi being the most aggressive pathogen to both oak species. The two most commonly isolated Phytophthora species, P. quercina and P. psychrophila, also proved their pathogenicity towards both Q. ilex and Q. faginea.