Plant Disease

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium ‘Candidatus Liberibacter spp.’, among which ‘Ca. L. africanus’ (CLaf) has posed a significant threat to citrus production in Africa for nearly a century. CLaf is closely related to the globally prevalent ‘Ca. L. asiaticus’ (CLas), whereas little is known about the virulence of CLaf, primarily because of limited genome resources. In this study, we completed the whole-genome assembly and annotation of the CLaf strain Zim (from Zimbabwe). Compared with CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effector (SDE) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that the CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of ‘Ca. L. solanacearum’, the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, V9J15_02010, V9J15_02015, V9J15_02025, and V9J15_02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

European blueberries (Vaccinium myrtillus L.) can be found across the Northern Hemisphere, particularly in cool, temperate forests. These shrubs produce dark blue berries that are rich in vitamins, antioxidants, and anthocyanins making them valuable for both human consumption and food supplements. Blueberries play a crucial ecological role as a food source for various animals (Pires et al., 2020). In Oct 2023, symptomatic plants were observed in the Aukštagiris Geomorphological Reserve (54°44′N.; 25°21′E.), Lithuania. These plants exhibited symptoms characteristic of phytoplasma infection, including reduced leaf size with a reddish color, witches’-broom structures, and stunted growth. Samples were collected from 5 asymptomatic and 20 symptomatic plants. The disease was designated as Vaccinium reddish witches'-broom (VacRWB). Total DNA was extracted from all samples and used as a template for nested PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 to amplify a 1.2 kb fragment of the phytoplasma 16S rRNA gene (Lee et al., 1998)., Primers Tuf340/Tuf890 and Tuf400/Tuf835 were used to amplify a 0.5 kb fragment of the phytoplasma elongation factor Tu (tuf) gene (Makarova et al., 2012). PCR amplicons were obtained from 18 symptomatic plants. Enzymatic RFLP analysis of the 1.2 kb 16S rDNA revealed that the phytoplasma is a member of 16SrVI-A and a ‘Candidatus Phytoplasma trifolii’-related strain. Three R16F2n/R16R2 and three Tuf400/Tuf835 amplicons were cloned, sequenced and were respectively identical. BLAST analysis of obtained sequences against the NCBI nt database showed 99.44% to 100% identities for 16S rRNA gene and 96.42% to 99.49% identities for tuf gene sequences compared to previously identified ‘Ca. P. trifolii’strains (first hits: PP237769 (matched 1250/1250 bp) and JQ824231 (389/391 bp), respectively). iPhyClassifier (Zhao et al., 2009) was used to generate in silico RFLP profiles of the 16S rDNA sequences, revealing an identical pattern (similarity coefficient = 1.00) to the reference pattern of subgruop 16SrVI-A (acc.: AY390261). Furthermore, the enzymatic and virtual RFLP profiles (using 14 endonucleases) matched each other. Phylogenetic analysis of the obtained VacRWB gene sequences confirmed the classification. Selected sequences were deposited in GenBank (NCBI) under Acc. nos PQ585819 (16S rRNA gene) and PQ594182 (tuf gene). Previously, a phytoplasma strain of group 16SrVI was reported in association with V. myrtillus plants in Austria (Borroto Fernandez et al., 2007). In Lithuania and other parts of Europe, infections by phytoplasmas of the 16SrIII-F subgroup are commonly associated with wild blueberries (Valiunas et al. 2004), often exhibiting slightly different symptoms (e.g., without reddish coloration and more severe stunting). To our knowledge, this is the first report of a ‘Ca. P. trifolii’-related strain infecting European blueberry in Northern Europe, including Lithuania. Recently, a ‘Ca. P. trifolii’-related strain (LingbSY) has been identified in lingonberries in Lithuania (Dėlkus et al., 2024). The susceptibility of both wild blueberries and lingonberries to related phytoplasma strains suggests the potential involvement of a shared insect vector, which warrants further investigations. These diseases pose a significant threat to forest ecosystems and could potentially spread to cultivated blueberries and lingonberries emphasizing the need for a comprehensive control strategy.

Guar or cluster bean (Cyamopsis tetragonoloba L.) is a leguminous crop well-suited for cultivation in arid and semi-arid regions. India accounts for 90% of world’s guar production. In September 2022, little leaf, and stunted plants (Fig. 1) were observed on 50 days-old guar plants with a disease incidence of less than 10%. To determine the cause, ten each of symptomatic (CyteJod1-10) and asymptomatic (CyteJod11-20) guar leaves were collected from the same field. Insects were also collected using the sweep-net method in polythene bags from in and around the guar fields. DNA extraction was done from leaf midribs of all the collected samples using the DNeasy Plant Mini Kit (Qiagen, Germany). Of the various insects collected(including aphids, whiteflies, thrips, mites, cutworms and leafhoppers) only the leaf hopper species are known to be putative vectors of phytoplasma (Ranebennur et al. 2022). Based on the morphological characters(De Long, 1931), the only leafhopper found within the field was Empoasca sp. which was further confirmed at a reference facility at the Division of Entomology, ICAR-IARI, New Delhi. Ten adults (Empoasca, C. tetragonoloba Jodhpur) were labelled (ECyteJod1-10) and used for DNA extraction using the Blood and Tissue kit (Qiagen, Germany).Nested polymerase chain reaction (Nested-PCR) was employed to determine phytoplasma species association using universal primers P1/P7 followed by R16F2n/R16R2 (Gundersen and Lee, 1996) and secA gene primers (secAfor1/secArev3 and secAfor2/secArev3) (Hodgetts et al., 2008). Out of the 10 symptomatic plants and 10 leaf hopper samples, 4 leaf hopper samples and all the symptomatic plants produced approximately 1.25 kb and 480 bp for the 16S rRNA and secA gene, respectively. The PCR products were cloned and sequenced. Since, all the 10 sequences were identical, only phytoplasma sequences of 2 isolates (CyteJod1& 2) were submitted to NCBI GenBank with accession numbers OQ058847-OQ058848 for 16S rRNA and OQ067519-OQ067520 for secA gene. Nucleotide BLAST analysis of 16S rRNA sequences revealed a 100% (1244/1244 nucleotides) identity with Ca. P. asteris (16SrI-B) obtained from leaf hopper (Tachycixius sp. Accession no. KF583778) from Lebanon, Italy. BLAST analysis of secA sequences revealed 100% (487/487 nucleotide) sequence similarity with 16SrI group of Ayapana triplinervis (water hemp plant) phytoplasma strain (OM142605) from India. The iPhyClassifier tool (Zhao et al., 2009) classified the 16S rRNA gene sequences into 16Sr group I, subgroup B, with a similarity coefficient of 1.0. Phylogenetic analysis done through MEGA11 (Tamura et al., 2021) showed that the guar phytoplasma strains clustered with 16SrI-B phytoplasma reference strains (Fig. 2a, 2b). The association of Empoasca sp. with 16SrI-B phytoplasma group has previously been reported in moth bean from Jodhpur, India (Singh et al., 2023). Previously, association of 16SrII-D subgroup with guar as host was reported from Haryana, India (Rao et al., 2019). This is a first report of guar disease caused by 16SrI-B subgroup phytoplasma worldwide. This has epidemiological significance and needs immediate attention since the pathogen may spread to other legume crops in similar geographical regions, posing a threat to agricultural systems where legumes are intercropped. The report highlights a new potential vulnerability for the guar and other pulses, emphasizing the need for vigilance and research to protect legume cultivation globally.

The Asian citrus psyllid (ACP) is the vector of Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening or Huanglongbing (HLB), one of the most devastating citrus diseases worldwide. The citrus industry in Georgia (U.S.A.) is in the process of a rapid expansion, and based on experiences with HLB in Florida, there is great concern about the potential impacts of HLB on this emerging industry. Prior to 2023, ACP had been identified in residential citrus trees in isolated Georgia counties but little to no testing of psyllids for CLas had occurred. However, in 2023, one individual psyllid collected from Chatham County was confirmed positive for CLas by PCR and sequencing. Furthermore, during 2023, ACP adults and nymphs were identified for the first time in a Georgia commercial citrus grove. The finding of ACP in a commercial planting represents a significant risk for CLas dissemination, and thereby has the potential to stall the rapid expansion of Georgia’s citrus industry. In the coming years, surveillance and testing of ACP from commercial groves will be essential for the early detection and management of HLB and its vector to reduce HLB spread within Georgia’s commercial groves.

‘Candidatus Phytoplasma brasiliense’ (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp., where it causes witches broom symptoms, and papaya, where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study, two quantitative PCR assays are developed: one using high-resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.

The occurrence of ‘Candidatus Liberibacter’ spp. and ‘Ca. Phytoplasma’ spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by qPCR. A set of primers and hydrolysis probes useful either in single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16S rDNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying ‘Ca. L. asiaticus’ (CLas), ‘Ca. L. americanus’ (CLam) and 16SrIX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the RNR test, though Cq values were higher when compared to 16S rDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cut-off established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.

Recurrent epiphytotics of X-disease, caused by ‘Candidatus Phytoplasma pruni,’ have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes and, more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multilocus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define “true” X-disease–inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extraorchard environment.

The phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb., and M. lucida [G.Forst.] Mabb.) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5′trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and quantitative real-time PCR (qPCR) on two to four occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded internal transcribed spacer (ITS) region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. To our knowledge, this is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine whether infections persist in the absence of D. citri.