Phytopathology Research

Abstract Citrus Huanglongbing (HLB) is one of the most devastating diseases in the citrus industry and is caused primarily by ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-restricted gram-negative bacterium transmitted via citrus psyllids in the field. Precise CLas detection is crucial for HLB control, particularly during extensive surveys in new emerging regions. Unfortunately, the lack of a practical on-site detection method for CLas due to the limited specificity of immunostrips and the costive non-user friendliness approaches prevent the effective disease control. In this study, a probe-based recombinase polymerase amplification (RPA) targeting the CLas five-copy nrdB gene (β-subunit of ribonucleotide reductase, RNR) was developed and evaluated for its specificity, sensitivity, and reliability. To enhance user-friendliness and facilitate widely use, an All-in-one kit was premade as freeze-dried pellets in individual tubes, containing all necessary components except DNA template. The applicability of the All-in-one kit for CLas detection was confirmed using plant or psyllid crude DNA extracts. Collectively, the All-in-one kit offers a specific, sensitive, rapid, cost-effective, and practical alternative for diagnosing CLas in field conditions when coupled with simplified crude DNA extraction method, providing an alternative approach for on-site CLas detection in the context of HLB.

Abstract Citrus Huanglongbing and Citrus tristeza are two diseases that affect the citrus industry worldwide. The pathogens causing these diseases are the phloem-limited bacteria ‘Candidatus Liberibacter spp.’ (mainly Ca. L. asiaticus, CLas) and citrus tristeza virus (CTV). We recently found that both CLas and CTV could be acquired and retained by the Asian citrus psyllid Diaphorina citri. However, the mechanism through which CLas and CTV interact with the insect vectors and plant hosts has not been defined. In this study, an electrical penetration graph was used to study the feeding behavior of D. citri adults on four groups of Citrus reticulata Blanco cv. Hongjü plants: healthy, CLas-infected, CTV-infected, and CTV-CLas coinfected plants. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) was applied to analyze the metabolites of the four groups of plants. The combined results are as follows: (1) The lowest number of metabolites were enriched in CTV-infected plants, which hardly influenced the feeding behavior of D. citri, suggesting that mild CTV strain (CT31) infection caused limited disorders in citrus plants compared with CLas infection; (2) Increased levels of L-arabinose and kaempferol in CTV-infected and CLas-CTV coinfected plants were suggested to contribute to increased penetration time during feeding of D. citri. CLas-infection increases the difficulty of finding appropriate feeding sites by the vector and results in xylem feeding for certain duration; (3) A significant reduction in α-linolenic acid metabolism in CLas-infected plants was found to be related to methyl jasmonate signaling, which induced resistance to D. citri and increased the duration of salivation. This effect was reversed by coinfection with CTV and was consistent with the phloem structure and carbohydrate accumulation alteration; (4) Stress response-associated 2'-hydroxygenistein and sakuranetin were highly upregulated flavonoid in CTV-CLas coinfected plants. This combinged with the anatomical alterations might interfere with D. citri feeding in the citrus phloem, as reflected by the time reduction of sap-sucking there. These findings will provide new insights into the interactions between CTV and CLas in citrus and the insect vector D. citri that transmiting these pathogens.

Abstract'Candidatus Phytoplasma solani’ (CPs), a phytoplasma endemic to the Euro-Mediterranean basin is a causative agent of several plant diseases, including the grapevine yellows disease “bois noir” (BN). As different CPs strains have been shown to have different ecological reservoirs and pathways for spread, the genetic characterization of CPs strains is a prerequisite, and better control of BN relies on the identification of reservoir plants. The variability of the phytoplasma genotypes involved in the BN pathosystem in Croatian vineyards was assessed by a multilocus sequence typing (MLST) approach. The genotyping was performed on selected grapevine, wild plants, and insects collected within the eleven years of national survey conducted in all Croatian viticultural regions. The extensive tuf, secY, stamp, and vmp1 genes-based MLST analyses revealed two new genotypes for stamp and vmp1 genes, designated as ST59 and V28, respectively, and overall identified 28 different CPs MLST genotypes. The prevalent MLST genotype in grapevine CPsSqt21 (S6/ST6/V18/tuf-b2) was widespread in nine counties across Uplands, Slavonia, and Danube wine regions and was affiliated to the known vector Hyalesthes obsoletus and to Urtica dioica. The other two most frequent genotypes were the U. dioica-associated CPsSqt28 (S39/ST46/V3/tuf-a) and the C. arvensis-associated CPsSqt2 (S1/ST9/V4/tuf-b1). CPs of different vmp1 genotypes was also detected in Cixius wagneri specimens originating from different parts of Croatia. In addition, CPs was detected in several Dichtyophara europaea insects and in two new potential plant reservoirs Ailanthus altissima and Robinia pseudoacacia. The substantial number of found MLST genotypes indicates the presence of several independent epidemiological cycles and is certainly a consequence of a unique geographical position of Croatia, bridging the different eco-climatic areas of central and south-eastern Europe.

AbstractIncreasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches’-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli-based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca. P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H2O2 accumulation in Nicotiana benthamiana, indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca. P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas.