Search results (224)


First Report of ‘Candidatus Phytoplasma aurantifolia’ Related Strain (16SrII-D) Associated with Stunting, Little Leaf and Phyllody Disease of Pearl Millet from South India

Citation
Hemalatha et al. (2022). Plant Disease
Names
Ca. Phytoplasma aurantifolia
Subjects
Agronomy and Crop Science Plant Science
Abstract
Pearl millet [Pennisetum glaucum (L).R.Br.] also known as bajra, is one of the oldest millets and is cultivated in dry regions of arid and semi-arid tropics where no other cereal can be successfully grown. Pearl millet cultivation in India accounts for about two-thirds of millet production and is the fourth most cultivated food crop after rice, wheat and maize in India (Reddy et al. 2021a). In February 2021, the typical symptoms of stunting, phyllody and little leaf were observed after 25-30 days after sowing pearl millet seeds at Agricultural Research Station in Perumallapalle, Tirupati, India (Fig.1 A-C). The disease incidence was recorded up to 20% in the sampling regions. Total DNA was extracted from two symptomatic and two asymptomatic plant samples using CTAB DNA extraction method (Murray and Thompson, 1980). The extracted DNA was amplified in direct PCR and nested PCR assay using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (Gundersen and Lee.1996) and secA gene with secAfor1/SecArev3 and SecAfor2/SecArev3 primer pairs (Hodgetts et al. 2008). 16SrRNA (1.25 kb) and secA (600 bp) gene amplicons were obtained from two symptomatic samples by nested PCR. No amplicons were produced with DNA from healthy leaf samples. Nested PCR amplified products (1.25 kb and 600 bp) from the symptomatic samples corresponding to the F2nR2 region of 16S rRNA and secA were directly sequenced at automated DNA sequencing facility (Eurofin Genomics India Pvt., Ltd Bangalore) and sequence data was deposited to NCBI GenBank with accession number ON005559 and ON067810. BLAST analysis revealed that pearl millet phytoplasma strain shared 100% sequence identity in 16Sr RNA and secA genes to ‘Canditatus Phytoplasma aurantifolia’ related strains (Acc. Nos. OM616883 and MT952965) from India. The subgroup was identified as 16SrII-D using the iPhyClassifier based on the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment (Zhao et al. 2009). The virtual RFLP pattern is similar to the reference pattern of 16SrII-D (Y10096) with similarity coefficient 1.00. Phylogenetic analysis of 16S rRNA and secA gene sequences using MEGA version 7.0 revealed that the pearl millet phytoplasma strain clustered with ‘Ca. P. aurantifolia’ isolates of 16SrII-D subgroup. (Fig.1D-E) Earlier, one of 16SrI-B-phytoplasma strain (HM 134245) associated with green ear disease of pearl millet was reported in North India (Kumar et al. 2010). In this study, we reported the association of 16SrII-D subgroup phytoplasma with little leaves and witches’-broom disease of pearl millet in South India. Phytoplasmas belonging to the 16SrII-D subgroup have a wide range of hosts, including the agricultural and horticultural crops (Reddy et al., 2021b). Hence, this is the first report of ‘Ca. P aurantifolia’ infection in bajra in South India. The increase in the spread of 16SrII-D sub group phytoplasma diseases and the expansion of the host range strongly suggest further studies on the epidemiology of the dynamic dissemination of this disease in India.

Physiological variables influenced by “<i>Candidatus</i> Liberibacter asiaticus” infection in two citrus species

Citation
Wu et al. (2022). Plant Disease
Names
Ca. Liberibacter asiaticus
Subjects
Agronomy and Crop Science Plant Science
Abstract
“Candidatus Liberibacter asiaticus” (CLas) is the bacterium associated with the citrus disease known as Huanglongbing (HLB). This study evaluated the influence of CLas infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers’ activity, and osmoregulation of two different species of citrus (the pomelo Citrus maxima (Burm.) Merr. and the mandarin C. reticulata cv. Tankan), relative to their measured CLas infection load. Results indicated all measured physiological variables excepting soluble sugar were affected by increased CLas infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. CLas infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with CLas scions revealed a significant decrease in the photosynthesis variables Fv/Fm, Y(II) and QL, whilst NPQ increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables SOD, CAT, POD, and soluble protein initially increased and later decreased. In addition, progression of CLas replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize the observed interspecific differences in physiological change are related to their relative resistance against CLas infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research: ultimately these detailed observations can facilitate the diagnosis of CLas infection.

An Improved Recombinase Polymerase Amplification Coupled with Lateral Flow Assay for Rapid Field Detection of ‘<i>Candidatus</i> Liberibacter asiaticus’

Citation
Rattner et al. (2022). Plant Disease
Names
Ca. Liberibacter asiaticus
Subjects
Agronomy and Crop Science Plant Science
Abstract
Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

First report of association of ‘<i>Candidatus</i> Phytoplasma asteris’ with <i>Moringa oleifera</i> leaf yellowing and stunting disease in India

Citation
Singh et al. (2022). Plant Disease
Names
Ca. Phytoplasma asteris
Subjects
Agronomy and Crop Science Plant Science
Abstract
Moringa oleifera (family Moringaceae) also known as the ‘drumstick tree’ is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture &amp; Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain ‘Candidatus Phytoplasma asteris’ (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with ‘Ca. Phytoplasma asteris’ (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B (‘Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.

Genome-Informed Design of a LAMP Assay for the Specific Detection of the Strain of ‘<i>Candidatus</i> Phytoplasma asteris’ Phytoplasma Occurring in Grapevines in South Africa

Citation
Alič et al. (2022). Plant Disease 106 (11)
Names
Ca. Phytoplasma asteri Ca. Phytoplasma
Subjects
Agronomy and Crop Science Plant Science
Abstract
Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid, and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific loop-mediated isothermal amplification (LAMP) assay for detection of a distinct strain of grapevine ‘Candidatus Phytoplasma asteris’ that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted the ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South Africa, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

First report of ‘Candidatus Phytoplasma asteris’ associated with witches'-broom disease of Cinnamomum camphora in China

Citation
Chen et al. (2022). Plant Disease
Names
Ca. Phytoplasma asteris
Subjects
Agronomy and Crop Science Plant Science
Abstract
Camphor tree (Cinnamomum camphora) has a wide distribution in the world and is mainly distributed in the South and southwest in China. It can be used as both a wood and a medicine, with high value in industry, medicine, and ecology. In May 2022, it was observed that the approximately ten to fifteen years old Camphor trees were exhibiting witches'-broom, small leaf morphology and chlorosis, and leaf drop in Panzhihua City, Sichuan Province, China. The witches'-broom symptoms consisted of many small branches with little leaves at the top of branches. It was named C. camphora witches'-broom disease (CCWB) and was found in some areas of Miyi, Yanbian, Renhe Xiqu and Dongqu counties. More than 28% of the plants were infected on the five areas surveyed. Total 100 samples were collected from five areas, with 15 symptomatic plants and 5 asymptomatic plants each area. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The nested PCR was employed to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions OP662614; OP662615; OP662616). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et al. 2003) (GenBank accessions OP649592; OP649593; OP649594). The fragment from 25 symptomatic samples was consistent with the positive control, and asymptomatic plants were negative, confirming an association of a phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CCWB phytoplasma showed that it has a 99.44% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.59% identity with rapeseed phyllody phytoplasma (GenBank accession CP055264). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of CCWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as ‘Ca. Phytoplasma asteris’-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CCWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the plants were positive for the phytoplasma using nested PCR after grafting for 30 d in nursery conditions. It is noteworthy that the plants were seriously damaged by aphid, Psyllidae and Ceroplastes. It is speculated that the insects of Homoptera typically transmit phytoplasmas by feeding on plant sap, thus it is necessary to control aphids in order to control the C. camphora witches'-broom disease. To the best of our knowledge, Camphor tree is a new host plant of ‘Ca. Phytoplasma asteris’ in China. The newly emerged disease is a threat to Camphor tree production.

First Report of ‘Candidatus Phytoplasma solani’ (16SrXII-A) Associated with Fox Nut (Euryale ferox Salisb.) Stolbur Disease in India

Citation
Biswas et al. (2022). Plant Disease
Names
Ca. Phytoplasma solani
Subjects
Agronomy and Crop Science Plant Science
Abstract
Fox nut also known as Gorgon nut, Makhana (Euryale ferox Salisb.) is a high value aquatic crop belonging to the family Nymphaeaceae. In India, it is generally grown in flood prone areas of North Bihar, lower Assam, parts of West Bengal, Odisha, Manipur and Tripura (Jana et al., 2018). India contributes nearly 70-80% of the global fox nut production. During September 2021, a phytoplasma like symptom was noticed on fox nut leaves at Basudeopur Farm of Research Centre for Makhana, Darbhanga, Bihar, India (23° 9' N and 65° 53' E). The characteristic symptom was that some portion of leaf lamina deformed along the veins with wrinkled and raised overgrowth or hypertrophy. The veins were thickened and reddened in the infected leaf area. The infection occurs in petiole as well as in flower stalk. The disease incidence was found as high as 30% which caused severe yield loss which was calculated to be 40% in that particular field. Total of 20 sampled fox nut plants, 10 symptomatic and 10 asymptomatic ones, were collected and tested for the presence of phytoplasma. A nested PCR assay using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (J. Jović et al. 2011) amplified the expected ~1.2-kb 16S rDNA fragment in all 10 symptomatic samples. No amplification was detected from asymptomatic samples. One of the ten 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced and was deposited in NCBI under the Accession no.OL873590. BLAST analysis showed that the sequence of the PCR 16S rDNA product was 100% identical to several GenBank sequences of Ca. P. solani (16SrXII Stolbur group) viz. KF907506. Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup. Identification of ‘Ca. P. solani’ was conducted following the STOL11 stolbur-specific protocol (Radonjić et al. 2009). Sequencing of tuf gene (Elongation factor Tu) was performed by using tuf marker genes (Cvrković et al. 2014) from 10 symptomatic and 10 asymptomatic samples. Sequence of the amplified gene (896 bp) was deposited in GenBank under Accession number OM174272. The presence of ‘Ca. P. solani’ was detected in all symptomatic samples, while all control plants tested negative. The RFLP analysis of tuf gene nested PCR products using HpaII endonuclease (Fermentas) revealed uniform tuf-b type in all positive samples. Nucleotide blast analyses showed that the tuf gene was 100% identical to STOL11 strain of C. P. solani subgroup 16SrXII-A (Accession No JQ797670). For developing a suitable management strategy, identification of the vector is essential. Leaf hoppers visiting the infected plants as well as nearby crop fields will be tested for presence of the phytoplasma. To the best of our knowledge, this is the first report of Candidatus Phytoplasma solani’ (16SrXII-A) infecting Fox nut (Euryale ferox Salisb.) in India. References Cvrković et al. 2014. Plant Pathol. 63:42. https://doi.org/10.1111/ppa.12080 Jana, B. R., et al. 2018. Int. J. Curr. Microbiol. App. Sci. 7(12): 578-587. https://doi.org/10.20546/ijcmas.2018.712.072 Jović, J. et al. 2011. B. Insectol. 64:S83. ISI Radonjić, S. et al. 2009. J. Phytopathol. 157:682. https://doi.org/10.1111/j.1439- 0434.2009.01560

First report of '<i>Candidatus</i> Phytoplasma palmae' (16SrIV-A subgroup) associated with palm Lethal Yellowing disease on <i>Cocos nucifera</i> and <i>Pritchardia sp.</i> in Guadeloupe, French West Indies

Citation
Pilet et al. (2022). Plant Disease
Names
Ca. Phytoplasma palmae
Subjects
Agronomy and Crop Science Plant Science
Abstract
Lethal Yellowing (LY) disease causes major damage to palms in Central America and the Caribbean. It has been reported as far south as Antigua (Myrie et al., 2014). LY affects over forty palm species, seriously impacts the coconut industry and alters the landscapes on islands with a tourist-based economy. In March 2021, the presence of LY disease was regularly monitored in Guadeloupe. Two palm species (Cocos nucifera and Pritchardia sp.) died on a private property in Saint-Anne, Grande Terre. Yellowing of lower fronds and necrosis of inflorescences were reported on some neighboring palms. One symptomatic Cocos nucifera (GP21-007) and four symptomatic Pritchardia sp. (GP21-005, GP21-006, GP21-008 and GP21-009) were sampled by stem drilling. Samples from four asymptomatic coconut trees (GP21-001 to GP21-004) were collected in the locality of Deshaies. DNA was extracted from the nine sawdust samples following a cetyltrimethylammonium bromide (CTAB) modified protocol (Doyle and Doyle, 1990). A quantitative polymerase chain reaction (PCR), following the protocol described by Christensen et al. (2004), was performed on DNA to diagnose the presence of phytoplasmas. An exponential amplification was observed for all DNA extracts from symptomatic palm samples (threshold number of PCR cycles (Ct) ranged from 18.50 to 23.58). DNA from asymptomatic samples yielded negative results (undetermined Ct). To identify the phytoplasma associated with LY, DNA samples were subjected to PCR, based on the 16SrRNA gene, plus internal transcribed spacers (ITS) using P1-1T (Pilet et al., 2021)/P7 (Schneider et al., 1995) primers, and secA gene using the primer pair secAFor1/secARev1 (Hodgetts et al. 2008). Amplicons of 1.8 kb covering the 16S ribosomal operon and 830 bp for the secA gene were produced using DNA from symptomatic trees. All amplicons were double strand sequenced (Genewiz, UK). The corresponding sequences were deposited in GenBank and subjected to BLASTn on NCBI. Sequences of the ribosomal operon gene (accession no. ON521114 to ON521118) were identical for the five positive samples. Sequencing revealed two distinct ribosomal operons with heterozygous peaks on the DNA chromatogram. The first aMino ambiguity (M = Adenine or Cytosine) was observed in the 16Sr RNA gene. The second was observed in the first intergenic transcript spacer. The 16S rDNA sequence (M = Cytosine) presented 100% identity with accession no. HQ613874 and 99.93% with accession no. U18747, the reference sequence for 'Candidatus Phytoplasma palmae'. The virtual RFLP pattern derived from the 16S rDNA F2nR2 fragment and identified using iPhyclassifier (Zhao et al. 2009) was identical to the reference pattern for the 16SrIV-A subgroup. A unique sequence was obtained for the partial secA gene (OP136139 to OP136143), sharing 100% identity with EU267187 for the palm LY phytoplasma preprotein translocase subunit (secA) gene. This is the first report of ‘Ca. Phytoplasma palmae’ (subgroup 16SrIV-A) associated with palm LY disease on Cocos nucifera and Pritchardia sp. in Guadeloupe. Measures to eradicate LY were implemented as soon as its presence was confirmed in Guadeloupe. LY phytoplasmas continue to spread in the Caribbean and are approaching South America, where the known vector, Haplaxius crudus, has already been reported (Silva et al., 2019). This poses a major threat to the coconut economy and the diversity of palm trees.