Plant Disease

The highly invasive field bindweed (Convolvulus arvensis), native to Europe and Asia, competes with crops, reduces yields, and acts as a reservoir for plant pathogens, increasing the potential for disease spread in agricultural systems (Sosnoskie et al. 2020; Wenninger et al. 2019). In July 2023, bindweed plants exhibiting little leaf, leaf discoloration, thickening, and overall stunted growth were observed in a 0.3 ha green cherry orchard in Kharja, Bani Kenanah, Irbid Governorate, Jordan (32°37'37.883"N, 35°52'49.185"E) (Fig. 1). Twenty out of 35 plants displayed symptoms, resulting in a 57% incidence rate. Since the symptoms resembled those associated with phytoplasma infections and bindweed's known role as a phytoplasma host (Jovic et al. 2021), DNA was extracted from six symptomatic plants and two healthy controls using the Qiagen DNeasy Plant Mini Kit, and PCR assay was conducted with the primer pair P1A/16S-SR to amplify near full-length phytoplasma 16S rDNA (about 1.5 kb) as described by Lee et al. (2004). All healthy controls tested negative, while all symptomatic plants tested positive. Amplicons were cloned into the pCRII-TOPO vector, sequenced, and a representative sequence was deposited in GenBank (Accession number PQ497569). Using iPhyClassifier (Zhao et al. 2009), the strain was identified as a 'Candidatus Phytoplasma omanense' (GenBank accession: EF666051, 16SrXXIX-A)-related strain, with 98.95% sequence identity, belonging to the 16SrXXIX group. The similarity coefficients were 0.93 and 0.97 compared to reference strains 16SrXXIX-A (EF666051) and 16SrXXIX-B (OL873126, Abu Alloush et al. 2023a), respectively. The phylogenetic analysis indicated that the newly detected field bindweed little leaf phytoplasma strain (designated FBLL1) is genetically distinct from these known subgroups and represents a novel subgroup, 16SrXXIX-C (Fig. 2). Further characterization of the FBLL1 strains was performed by PCR amplification of the rp (1.9 kb) and secY (1.3 kb) gene segments using specially designed primers: FBRP-F2/FBRP-R4 (5’-TTGGCCGCCTTCAAATCCTA-3’/5’-TCGGAGGAGAAGTTTTGGCT-3') for rp, and FBSecYF1/FBSecR1 (5’-CTTCTTTTGGTGATATCCCA-3’/5’-TGGCGGAAGTATTGAGATAAGAA-3') for secY. The resulting rp and secY gene sequences were deposited in GenBank (Accession numbers PQ505136 and PQ505137). BLASTN analysis revealed that the rp gene's top hit was 'Ca. P. pruni' (CP119306) with 75.60% identity, while the secY gene's top hit was 'Ca. P. phoenicium' (MN076652), with 73.47% identity. Compared to the 16S rRNA gene, the lower percent identity is due to the limited availability of 16SrXXIX rp and secY gene sequences for comparison. Field bindweed hosts several phytoplasmas, such as ‘Ca. P. solani’ (16SrXII-A), which is linked to Bois noir (BN) in grapevines, and ‘Ca. P. convolvuli’ (16SrXII-H), associated with bindweed yellows (Jovic et al. 2021; Abu Alloush et al. 2023b). ‘Ca. P. omanense’ has also been documented in bindweed in Lebanon (Fossaic et al. 2019) and Iran (Esmailzadeh Hosseini et al. 2016). As a confirmed reservoir of the BN phytoplasma, bindweed supports the pathogen’s persistence and vector-mediated transmission to grapevines (Jovic et al. 2021). This report marks the first identification of ‘Ca. P. omanense’-related strains (16SrXXIX-C, novel subgroup) in bindweed in Jordan. These findings suggest that bindweed may serve as a potential reservoir for a broader range of phytoplasmas than previously recognized, with a wider geographical distribution. Its invasive growth near crops, such as grapevines, raises concerns about the potential phytoplasma spillover. This highlights the need for improved monitoring and management, which includes targeted herbicide application, mechanical removal, and biological control to protect agricultural biosecurity.

Lethal wilt (LW), also known as “Marchitez Letal (ML)” in Colombia, is an endemic disease affecting oil palms (Elaeis guineensis Jacq.) and is a leading cause of crop loss. The disease is characterized by the drying of leaflets from the tip to the base, primarily impacting the lower third of the plant and progressively moving upward. This progression leads to physiological disturbances, including necrosis at the tips of immature inflorescence bracts (spines) and the detachment of bunch fruits, ultimately causing wilting. As a phytosanitary measure, infected palms are eradicated to prevent further spread of the disease. The primary goal of this research was to identify the bacteria associated with LW and to validate a molecular detection method. A 16S amplicon-based analysis was employed to identify and compare the microbial diversity in LW-affected palm tissues with those of healthy plants. Among the 16 OTUs corresponding to different bacterial genera found in all LW samples, taxonomic classification and symptomatology suggested that the bacteria closely associated with LW belong to the genus Candidatus Liberibacter. Further phylogenetic analysis indicated that these bacteria are part of the Rhizobiaceae family, grouping closely with other species of the genus Candidatus. Liberibacter. The concentration of the pathogen in different oil palm tissues was determined using droplet digital PCR (ddPCR) and quantitative PCR (qPCR), expressed in copies/µL in the LW samples. This study represents the first report of ‘Candidatus Liberibacter sp’. being associated with lethal wilt in oil palms of the Arecaceae family in Colombia. The findings from this research have the potential to contribute significantly to the development of effective management strategies to prevent crop losses.

Surveys for exotic plant pests conducted during July and August of 2023 and 2024 across 20 vineyards in 12 counties throughout Minnesota, USA, revealed that less than 2% of the approximatively 3000 vines inspected (Vitis spp., hybrid grape varieties) exhibited symptoms suggestive of phytoplasma yellows disease. Observed symptoms included yellowing of leaf lamina, downward rolling of leaf margins, and necrosis of leaf margins. To investigate a potential association between these symptoms and phytoplasmas, two symptomatic plants were selected in August of 2024 from two distinct vineyards for further analysis. Petiole tissue DNA extracts were initially tested using a phytoplasma-specific real-time PCR assay (Hodgetts et al. 2009), which confirmed the presence of phytoplasma DNA in both symptomatic plants. Subsequently, the samples underwent semi-nested PCR amplification targeting the phytoplasma 16S rRNA gene, using primers P1/16S-SR followed by P1A/16S-SR (Deng and Hiruki 1991; Lee et al. 2004). The resulting amplicons were cloned and sequenced. No amplicon was produced from an asymptomatic grapevine DNA sample included as a test control. Analysis of the 16S rRNA gene sequences revealed that each plant was infected by one of two distinct phytoplasma strains, designated MN450 and MN466. Representative sequences obtained from the two samples exhibiting interoperon heterogeneity were deposited in the GenBank database under accession numbers: PQ889195 (rrnA) and PQ889196 (rrnB) for strain MN450, and PQ889197 (rrnA) and PQ889198 (rrnB), for strain MN466. Sequence analysis using the online phytoplasma classification tool iPhyClassifier (Zhao et al. 2009) showed that the 16S rRNA gene sequences from MN450 shared 1491/1495 bp (99.73%) and 1492/1495 bp (99.80%) identity (rrnA and rrnB, respectively) with that of the ‘'Ca. Phytoplasma pruni' rrnA’ reference strain (GenBank JQ044393), identifying MN450 as a ‘'Ca. Phytoplasma pruni' rrnA’-related strain and possibly representing a new subgroup within the 16Sr group III. For MN466, the 16S rRNA gene sequences shared 1471/1482 bp (99.26%) and 1469/1482 bp (99.12%) identity (rrnA and rrnB, respectively) with that of the ‘Ca. Phytoplasma asteris’ reference strain (GenBank M30790), and identifying the phytoplasma as a ‘Ca. P. asteris’-related strain belonging to subgroup 16SrI-A. To further characterize the two phytoplasma strains, the secY gene was PCR amplified from the DNA extracts as described by Lee et al. (2010). The obtained amplicons were cloned and sequenced, and representative secY gene sequences were deposited in GenBank under accession numbers PQ899621 (MN450) and PQ899622 (MN466). BLASTn searches against the NCBI core nucleotide database revealed that the secY gene sequence for MN450 exhibited 100% identity (1614/1614 bp) with ‘Ca. P. pruni’ (GenBank KM268860). Similarly, the sequence for MN466 showed 100% identity (1287/1287 bp) with ‘Ca. P. asteris’ (GenBank CP000061). In North America, grapevine yellows disease associated with strains related to 'Ca. P. asteris' and 'Ca. P. pruni' has previously been reported in various states and referred to as North American grapevine yellows (NAGY) by Davis et al. (2015). To our knowledge, this is the first report of NAGY in Minnesota, confirming that the two phytoplasma species detected are consistently associated with the disease in U.S. vineyards. While further research is needed to evaluate the potential impacts of NAGY in Minnesota, this study highlights the broader distribution of the disease across the United States.

‘Candidatus Liberibacter asiaticus’ (CLas), the agent associated with the Huanglongbing (HLB) citrus disease, is commonly detected using quantitative polymerase chain reaction (qPCR) with hydrolysis probes. Internal standards are typically included in the qPCR assays to reduce the risk of false negatives caused by inhibitors. When the internal standard is detected but CLas is not, it is generally assumed that the pathogen is absent from the tested sample. However, our study shows that trace amounts of CLas may go undetected if the internal standard is either overly abundant or too dissimilar to CLas. To overcome these limitations, we developed a synthetic internal standard (IS) that uses the same primer as the CLas target sequence, along with three to four downstream nucleotides, but with a unique internal sequence derived from smooth hammerhead shark (Sphyrna zygaena; IS-SHK). This sequence matches the G/C content and melting temperature of the CLas target and was specifically selected to minimize potential interference from other nucleic acid materials in citrus samples. To minimize competition between the IS-SHK standard and the CLas target, an average of only 21 molecules of IS-SHK standard is added to each qPCR reaction. Therefore, when IS-SHK standard is detected at expected levels and CLas is not, the absence of CLas is confirmed rather than inhibition of the detection. Conversely, the absence of both IS-SHK standard and CLas suggests the presence of a qPCR inhibitor, warranting retesting of the sample.

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 quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either 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 16Sr DNA 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 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA 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 cutoff 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.

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.

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.