Agronomy and Crop Science

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by ‘ Candidatus Liberibacter asiaticus’ ( CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.

Areca catechu palm is an important cash plant in Hainan Island of China and also in the tropical regions of the world. A. catechu palm yellow leaf (AcYL) disease caused by phytoplasmas is a devastating disease for plant production. In the study, the phytoplasmas associated with the AcYL disease were identified and characterized based on their conserved genes, and genetic variation and phylogenetic relationship of the phytoplasma strains in the 16SrXXXII group were demonstrated. The results indicated that A. catechu palm plants showing yellow leaf symptoms were infected by ‘Candidatus Phytoplasma malaysianum’-related strains belonging to the 16SrXXXII-D subgroup. BLAST and multiple sequence alignment analysis based on 16S rRNA and secA genes showed that the AcYL phytoplasmas shared 100% sequence identity and 100% homology with the ‘Ca. P. malaysianum’-related strains. Phylogenetic analysis indicated that the AcYL phytoplasmas and ‘Ca. P. malaysianum’-related strains belonging to the 16SrXXXII group clustered into one clade with a 100% bootstrap value. Based on computer-simulated digestions, six kinds of restriction fragment length polymorphism patterns within the 16SrXXXII group were obtained, and a novel subgroup in the 16Sr group was recommended to propose and describe the relevant strains in this 16Sr subgroup. To our knowledge, this is the first study to report that A. catechu palm showing yellow leaf symptoms was infected by ‘Ca. P. malaysianum’-related strains belonging to the 16SrXXXII group. A novel 16Sr subgroup, 16SrXXXII-F, was proposed based on the systematical analysis of genetic variation of all phytoplasmas within the 16SrXXXII group. The findings of this study will support references for monitoring the epidemiology and developing effective prevention strategies for AcYL disease.

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

Huanglongbing (HLB) is a citrus infectious disease caused by ‘Candidatus Liberibacter’ spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3′-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of ‘Candidatus Liberibacter asiaticus’ (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

‘Candidatus Liberibacter’ spp. are the most prevalent microorganisms in the citrus plant, associated with citrus huanglongbing, which are transmitted by psyllid vectors. In Colombia, the vector Diaphorina citri Kuwayama has been reported in different regions, but ‘Ca. Liberibacter asiaticus’ (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1/OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a/CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we used droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of the ribonucleotide reductase gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country’s disease management, control strategies, and general crop health. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Citrus greening disease was first reported in Saudi Arabia during the 1970s when characteristic foliar and fruit symptoms were observed in commercial citrus groves. However, ‘Candidatus Liberibacter asiaticus’ (CLas) was not detected in symptomatic trees until 1981 to 1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the 13 citrus-growing regions of the country. Citrus trees were surveyed for the presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults revealed that trees were positive for CLas detection in 10 of the 13 survey regions; however, CLas was undetectable in ACP adults. Phylogenetic and single nucleotide polymorphism (SNP) analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (∼1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, U.S.A., isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.