Plant Disease

Areca palm yellow leaf phytoplasma (AYLP) and Areca palm velarivirus 1 (APV1) are two critical pathogens associated with yellow leaf in areca palm, resulting in devastating damage to areca production. However, evidence of their co-infection remains unclear. Areca palms showing yellow leaf symptoms (suspected to be caused by phytoplasma and velarivirus 1) were surveyed and sampled around Hainan Island of China and the pathogens were identified and analyzed in the study. The results showed that infectious leaf yellowing symptoms of areca palms primarily occurred in eastern and southern cities and counties of Hainan Island. Molecular identification revealed co-infection of 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) and APV1 in individual areca palm samples. Among 520 samples tested by qPCR, AYLP was detected in 23.46% (n=122), APV1 in 53.46% (n=278), and a co-infection with both AYLP and APV1 in 10.96% (n=57). A statistically significant difference in APV1 detection rate was observed between asymptomatic and symptomatic plantations (P < 0.0001), suggesting APV1 infection likely represents an important risk factor for yellowing development in cultivated areca palm populations. These results confirm the co-infection of AYLP and APV1 on Hainan Island, offering epidemiological evidence for targeted disease management.

Pulasan (Nephelium mutabile Blume; family Sapindaceae) is a tropical fruit known for its high orilagin, ascorbic acid and thiamine content, known for its potent antioxidant and anti-inflammatory properties. Native to Southeast Asia, pulasan is extensively cultivated in Malaysia, Thailand, Indonesia, Sri Lanka, India and Philippines (Afzaal et al., 2023). In India, pulasan is cultivated in tropics, where it is cherished as a homestead crop. A recent survey conducted in September 2024 at Pangode village (8°45'23.22"N 76°58'48.68"E), Thiruvananthapuram, Kerala, showed little leaf symptoms along with drying of side branches along with stunted growth in pulasan trees (variety P15). Only two pulasan trees were recorded with symptoms out of 20 trees examined in the campus. To investigate the molecular identity of the phytoplasma, leaf samples from two symptomatic and asymptomatic trees were collected and subjected to total DNA extraction by DNA extraction kit (Qiazen). The extracted DNA was amplified using nested PCR assays with universal primers P1/Tint (Smart et al., 1996) followed by 3Fwd/3Rev (Manimekalai et al., 2010) specific for the 16S rRNA gene and tuf gene-specific primer pairs fTufl/rTufl (Schneider et al., 1997). The PCR assays yielded amplicons of ~1.3 kb and ~1.1 kb for the 16S rRNA and tuf genes, respectively, from all the symptomatic leaf samples, whereas no amplification was observed from any asymptomatic samples. One sequence of each gene of pulasan phytoplasma strain (PK-1) were deposited into the GenBank database [(Acc Nos. PQ865399 (16S rRNA gene) and PQ877893 (tuf gene)]. BLASTn analysis of the both gene sequences of the PK strains revealed a 99.62% sequence identity with eggplant little leaf (Acc. No. MW273757; 16S rRNA gene) and 100% sequence similarity with Cucurbita pepo phytoplasma (Acc. No. KX358589; tuf gene) belongs to the clover proliferation phytoplasma subgroup. Phylogenetic analysis using MEGA 11 employing the Maximum likelihood method, demonstrated that the PK phytoplasma strains clustered within the 16SrVI-D subgroup-related strains. The virtual RFLP pattern generated for the 16S rRNA gene of PK phytoplasma strain through pDRAW analysis was found identical to the reference pattern of 16VI-D subgroup (Periwinkle little leaf phytoplasma; GenBank Acc. No. AF228053). Sequence comparison analysis confirmed the presence of a 'Candidatus Phytoplasma trifolii'-related strain associated with pulasan showing little leaf disease and stunting in India. While, phytoplasma associations have been reported in various fruit species globally (Fiore et al., 2018; Mitra et al., 2023), this study represents the first documented case of a phytoplasma disease infecting pulasan worldwide. Notably, 'Ca. P. trifolii' (16SrVI-D subgroup) has previously been identified associated with several economically important agricultural and horticultural crops worldwide (Zhao and Wei, 2020; Rao, 2021). The results of this study revealed that pulasan trees serve as an alternative natural reservoir for the ‘Candidatus Phytoplasma trifolii’ (16SrVI-D) phytoplasma strain, may potentially facilitate its dissemination to other crops through insect vectors. Considering the significant phytosanitary concerns, epidemiological factors and economic impact of pulasan in Southeast Asia for its global export potential, the detection of phytoplasma-associated disease in pulasan is noteworthy as it not only poses a potential threat to cultivation and productivity but may also have broader implications for regional trade and biosecurity protocols.

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.

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 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.