First Report of 'Candidatus Phytoplasma asteris'-related Strain (16SrI group) Associated with Small Leaves, Leaf Yellowing, and Shoot Proliferation of Morning Glory (Ipomoea biflora) in Taiwan.

Ipomoea biflora L., commonly known as morning glory, is an herbaceous vine plant in the Convolvulaceae family and is widespread at low elevations in Taiwan and other East Asian countries. In September 2023, six I. biflora plants exhibiting small leaves, leaf yellowing, and shoot proliferation were observed in a vacant lot in Taiwan Agricultural Research Institute (TARI), Wufeng District, Taichung, Taiwan, representing 100% disease incidence in the area. All the symptomatic morning glory climbed onto Murraya paniculata L. (common jasmine orange) which however showed no similar symptoms. The total DNA (two samples for each plant) from leaf tissues of three symptomatic morning glory plants, two asymptomatic morning glory plants, and one asymptomatic common jasmine orange was isolated by the CTAB method (Fulton et al. 1995) and used for PCR with the universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995), to amplify a fragment containing partial 16S rDNA. Expected 1.8-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no PCR product was detected from that of the asymptomatic I.biflora and M. paniculata plants. Six PCR products were cloned and sequenced in the Biotechnology Center DNA-sequencing facility at National Chung Hsing University, and one representative sequence was selected and deposited in GenBank. BLAST analysis revealed that the obtained 16S rDNA sequence (PP230905) shared 99.92% identity with the following phytoplasma strains: rapeseed phyllody phytoplasma (CP055264), plumbago auriculata leaf yellowing phytoplasma (MN239503), and aster yellows phytoplasma (MK992774), which all belong to the 16SrI subgroup. The query 16S rDNA sequence shares 99.84% identity with that of the 'Candidatus Phytoplasma asteris' reference strain (M30790), suggesting that the phytoplasma is a 'Ca. Phytoplasma asteris'-related strain. A virtual restriction fragment length polymorphism (RFLP) analysis was conducted using iPhyClassifier tool (Zhao et al. 2009), and the pattern derived from the 16S rDNA fragment of the I. biflora phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of 16SrI, subgroup B (onion yellows phytoplasma OY-M; AP006628). Six total DNA samples from symptomatic plants were used as templates to amplify 842 bp secA sequences with SecAfor1 and SecArev3 primers (Hodgetts et al. 2008), and one representative sequence was deposited in GenBank. The partial secA sequence (PP263636) showed 98.22% identity with that of Trema levigatum witches'-broom phytoplasma (MW032212) that also belongs to the 16SrI group (Wan et al. 2021). Phylogenetic analysis of both 16S rDNA and secA confirmed I. biflora phytoplasma as 16SrI, subgroup B. Taken together, we concluded that the morning glory phytoplasma in this study was a 'Ca. Phytoplasma asteris'-related strain belonging to the 16SrI group. To the best of our knowledge, this is the first report of a phytoplasma-infected I. biflora in Taiwan, suggesting morning glory as a new natural host of 16SrI phytoplasmas, alongside other plants like roselle and citrus (Tseng et al. 2014; Feng et al. 2015).

Insecticidal action of mammalian galectin-1-transfected Arabidopsis thaliana.

BACKGROUND: Galectins (GALs) are a family of mammalian sugar-binding proteins specific for ß-galactosides. Our previous studies have shown that the larval development of the diamondback moth (Plutella xylostella) is significantly disturbed when fed with recombinant mammalian galectin 1 (GAL1) derived from Escherichia coli. To further explore its applicability, two GAL1-overexpressed Arabidopsis [GAL1-Arabidopsis (whole plant) and GAL1-Arabidopsis-vas (vascular bundle-specific)] lines were established for insecticidal activity and mechanism studies. RESULTS: The expression level of GAL1 in transgenic Arabidopsis is 1-0.5% (GAL1-Arabidopsis) and 0.08-0.01% (GAL1-Arabidopsis-vas) of total leaf soluble protein. Survival, body weight, and food consumption significantly decreased in a time-dependent manner in P. xylostella larvae (with chewing mouthparts) fed on GAL1-Arabidopsis. The mortality of Kolla paulula (with piercing-sucking mouthparts and xylem feeder) fed on GAL1-Arabidopsis-vas was also significantly higher than that fed on wild-type Arabidopsis (WT-Arabidopsis), but was lower than that fed on GAL1-Arabidopsis. The histochemical structure and results of immunostaining suggested that the binding of GAL1 to the midgut epithelium of P. xylostella fed on GAL1-Arabidopsis was dose- and time-dependent. Ultrastructural studies further showed the disruption of microvilli, abnormalities in epithelial cells, and fragments of the peritrophic membrane (PM) in P. xylostella larvae fed on GAL1-Arabidopsis. CONCLUSION: The insecticidal mechanism of GAL1 involves interference with PM integrity and suggests that GAL1 is a potential candidate for bioinsecticide development. © 2024 Society of Chemical Industry.

TaqMan Quantitative PCR Detection of Xylella taiwanensis in Taiwan.

Xylella taiwanensis (Xt) is a nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, loss of tree vigor, and reduction in fruit yield and quality. No cure for PLSD is available. The only option for growers to control the disease is to use pathogen-free propagation material, which requires early and accurate detection of Xt. Currently, only one simplex PCR method is available for the diagnosis of PLSD. We developed five Xt-specific TaqMan quantitative PCR (TaqMan qPCR) systems (primers-probe sets) for the detection of Xt. The PCR systems target three conserved genomic loci commonly used in bacterial pathogen detection: the 16S rRNA gene (rrs), the 16S-23S rRNA intergenic transcribed sequence (16S-23S rRNA ITS), and the DNA gyrase gene (gyrB). BLAST analysis using the GenBank nr sequence database, including whole genome sequences of 88 Xanthomonas campestris pv. campestris (Xcc) strains, 147 X. fastidiosa (Xf) strains, and 32 Xt strains, showed that all primer and probe sequences were specific only to Xt. Single nucleotide polymorphisms (SNPs) provided the primer/probe specificity to Xt. The PCR systems were evaluated by using DNA samples from pure cultures of two Xt strains, one Xf strain, one Xcc strain, and 140 plant samples collected from 23 pear orchards in four counties in Taiwan. The two-copy rrs and 16S-23S rRNA ITS-based PCR systems (Xt803-F/R, Xt731-F/R, and Xt16S-F/R) showed higher detection sensitivity than the two single-copy gyrB-based systems (XtgB1-F/R and XtgB2-F/R). A metagenomic analysis of a representative PLSD leaf sample detected the presence of non-Xt proteobacteria and fungal pathogens that should be taken into consideration in PLSD, as they might interfere with diagnosis.

The Bph45 Gene Confers Resistance against Brown Planthopper in Rice by Reducing the Production of Limonene.

Brown planthopper (BPH), a monophagous phloem feeder, consumes a large amount of photoassimilates in rice and causes wilting. A near-isogenic line 'TNG71-Bph45' was developed from the Oryza sativa japonica variety 'Tainung 71 (TNG71) carrying a dominant BPH-resistance locus derived from Oryza nivara (IRGC 102165) near the centromere of chromosome 4. We compared the NIL (TNG71-Bph45) and the recurrent parent to explore how the Bph45 gene confers BPH resistance. We found that TNG71-Bph45 is less attractive to BPH at least partially because it produces less limonene. Chiral analysis revealed that the major form of limonene in both rice lines was the L-form. However, both L- and D-limonene attracted BPH when applied exogenously to TNG71-Bph45 rice. The transcript amounts of limonene synthase were significantly higher in TNG71 than in TNG71-Bph45 and were induced by BPH infestation only in the former. Introgression of the Bph45 gene into another japonica variety, Tainan 11, also resulted in a low limonene content. Moreover, several dominantly acting BPH resistance genes introduced into the BPH-sensitive IR24 line compromised its limonene-producing ability and concurrently decreased its attractiveness to BPH. These observations suggest that reducing limonene production may be a common resistance strategy against BPH in rice.

Do Sharpshooters From Around the World Produce the Same EPG Waveforms? Comparison of Waveform Libraries From Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) Vectors Kolla paulula (Hemiptera: Cicadellidae) From Taiwan and Graphocephala atropunctata From California.

When an exotic invasive species is a vector-borne plant pathogen, vector feeding behavior must be studied to identify potential host plant range and performance of specialized pathogen transmission behaviors. The most rigorous tool for assessing vector feeding behavior is electropenetrography (EPG). Xylella fastidiosa Wells et al. is a gram-negative bacterium native to the Americas, where it is the causal agent of lethal scorch-type diseases such as Pierce's disease (PD) of grapevines. In 2002, a PD strain of X. fastidiosa invaded Asia for the first time, as confirmed from grape vineyards in Taiwan. Kolla paulula (Wallker), a native Asian species of sharpshooter leafhopper, was found to be the primary vector in Taiwanese vineyards. This study used an AC-DC electropenetrograph to record stylet probing behaviors of K. paulula on healthy grapevines. The main objective was to create an EPG waveform library for K. paulula. Waveform description, characterization of R versus emf components (electrical origins), and proposed biological meanings of K. paulula waveforms are reported. In addition, comparison of K. paulula waveforms with those from the most efficient, native vector of X. fastidiosa in California vineyards, Graphocephala atropunctata, is also reported. Overall, both species of sharpshooters had similar-appearing waveforms. Five new findings were identified, especially that the previously described but rare waveform subtype, B1p, was extensively produced in K. paulula recordings. Sharpshooter waveforms from species worldwide share a high degree of similarity. Thus, EPG methods can be rapidly applied to potential vectors where X. fastidiosa is newly introduced.

Review of the EPG Waveforms of Sharpshooters and Spittlebugs Including Their Biological Meanings in Relation to Transmission of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae).

Electropenetrography (EPG) is one of the most rigorous methods to study stylet probing behaviors of piercing-sucking insects whose mouthparts move invisibly inside hosts. EPG is particularly useful for identifying vector behaviors that control transmission (acquisition, retention, and inoculation) of plant pathogens, comparing those behaviors among vector species, and aiding in development of novel vector and disease management tactics. Xylella fastidiosa (Wells et al.) is a gram-negative, invasive bacterium native to the Americas, where it is the causal agent of lethal scorch-type diseases such as Pierce's disease of grapevines. Xylella fastidiosa is transmitted by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and spittlebugs (Hemiptera: Aphrophoridae). Despite over 75 yr of study, details of the inoculation mechanism of X. fastidiosa were unknown until the advent of EPG research with sharpshooters. Herein, the following topics are presented: 1) review of key EPG principles and waveforms published to date, emphasizing sharpshooters and spittlebugs; 2) summary of present understanding of biological meanings of sharpshooter waveforms; 3) review of mechanisms of transmission for X. fastidiosa illuminated by EPG; and 4) recommendations of the most useful waveform categories for EPG use in future, quantitative comparisons of sharpshooter stylet probing on various treatments such as infected versus uninfected plants, resistant varieties, or insecticide treatments. In addition, new work on the functional anatomy of the precibarial valve is discussed in the context of X. fastidiosa transmission and EPG waveforms. Also, the first block diagram of secondary, signal-processing circuits for the AC-DC EPG is published, and is discussed in relation to EPG signals appearances and meanings.

Draft Genome Sequence of "Candidatus Sulcia muelleri" Strain KPTW1 from Kolla paulula, a Vector of Xylella fastidiosa Causing Pierce's Disease of Grapevine in Taiwan.

The genome of "Candidatus Sulcia muelleri" strain KPTW1 from Kolla paulula, a vector of Xylella fastidiosa that causes Pierce's disease (PD) of grapevine in Taiwan, was sequenced. The strain has a genome size of 253,942 bp, GC content of 22.7%, 237 predicted protein-coding genes, and 34 RNA genes.

Transgenic plants that express the phytoplasma effector SAP11 show altered phosphate starvation and defense responses.

Phytoplasmas have the smallest genome among bacteria and lack many essential genes required for biosynthetic and metabolic functions, making them unculturable, phloem-limited plant pathogens. In this study, we observed that transgenic Arabidopsis (Arabidopsis thaliana) expressing the secreted Aster Yellows phytoplasma strain Witches' Broom protein11 shows an altered root architecture, similarly to the disease symptoms of phytoplasma-infected plants, by forming hairy roots. This morphological change is paralleled by an accumulation of cellular phosphate (Pi) and an increase in the expression levels of Pi starvation-induced genes and microRNAs. In addition to the Pi starvation responses, we found that secreted Aster Yellows phytoplasma strain Witches' Broom protein11 suppresses salicylic acid-mediated defense responses and enhances the growth of a bacterial pathogen. These results contribute to an improved understanding of the role of phytoplasma effector SAP11 and provide new insights for understanding the molecular basis of plant-pathogen interactions.