Detection of Asian Citrus Psyllid (Hemiptera: Psyllidae) in Ethiopia: A New Haplotype and its Implication to the Proliferation of Huanglongbing

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid, is a pest of citrus known for its transmission of Candidatus Liberibacter asiaticus (Ca. L. asiaticus), the causal bacterium of Huanglongbing. The African citrus triozid Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) has been the putative vector of Candidatus Liberibacter africanus (Ca. L. africanus) which causes the African citrus greening disease, until the recent detection of D. citri on the continent. Following reports of D. citri in Kenya and Tanzania, we surveyed citrus plants to establish the presence/absence of D. citri in Ethiopia in citrus-growing regions ranging from 900 to 2,460 m above sea level (masl). Diaphorina citri adults were detected in five of the surveyed sites in Ethiopia. Adult insects encountered were collected using an aspirator and stored in 97% ethanol. The mitochondrial cytochrome oxidase 1 (mt COI) gene of the collected insects was amplified using LepF1/LepR1 primers, and sequences obtained showed low variation, which fell within the acceptable range of species. BLAST was used to query the sequences obtained, and all the sequences linked to D. citri accessions that are available in GenBank. The analysis of the sequences revealed a new haplotype of the species that differs from haplotypes previously reported. Phylogenetic relationships of our samples and other D. citri reference sequences was inferred using the Maximum-likelihood method. Monophyly was observed between the samples and the publicly available sequences from global accessions. This is the first report of the presence of D. citri in Ethiopia.

Complete Genome Sequence of Indian Race 4 of Xanthomonas oryzae pv. oryzae, the Causal Agent of Bacterial Blight of Rice

Xanthomonas oryzae pv. oryzae, the causal bacterium of bacterial blight limits rice production globally. Currently, genome sequences for only a few X. oryzae pv. oryzae isolates are available from India. Based on the next-generation sequencing and single-molecule sequencing in real-time technologies, we present here the complete genome sequence of X. oryzae pv. oryzae race 4, a highly virulent member of the Indian X. oryzae pv. oryzae population that has been extensively used in different research studies. The genome data will contribute to our understanding of X. oryzae pv. oryzae genomic features and pave the way for research on rice–X. oryzae pv. oryzae interactions.

Identification of bacterial pathogen associated with red stripe/top rot disease of sugarcane in Punjab, India

This study was aim to identify the pathogen associated with the red stripe/top rot disease. The isolates were collected from major sugarcane growing districts of Punjab. Molecular identification of the pathogen was carried out to confirm the associated organism with this disease. Of the different sets of specific PCR based molecular markers were used, primer sets SeQ1 (Aaa) and SeQ2 (Aaa) amplified the expected 550bp of ITS (Internal Transcribed Spacer) region of the rDNA which revealed the pathogen as Acidovorax avenae pv. avenae, whereas the primer set Aaaf3 and Aaar2 are specific to strains that infect rice crop did not amplify any fragment. Our studies confirmed Acidovorax avenae pv. avenae as the causal bacterium associated with the red stripe/top rot of sugarcane in Punjab.

Fruit Rot of Tinda Caused by Pseudomonas aeruginosa–A New Report from India

Fruit rot disease (FRD), an emerging problem of tinda (Praecitrullus fistulosus) in India. FRD epidemics begin during rainy and warm weather and often spoil marketable produce. Symptoms appear as numerous, pale brown-to-dark brown, deeply penetrating circular soft rot lesions on fleshy fruit tissues. Noneffervescent bacterial exudates occasionally form on lesions. Repeated isolations from FRD-affected tinda fruits consistently yielded the same bacterial species. Inoculation of the isolated bacterium into asymptomatic tinda fruits produced identical soft rot symptoms. Fruits were inoculated with the isolate ITCC B0030 (0.1 OD) by removing a 2.0-cm deep tissue plug with a sterile cork borer (5 mm in diameter) and injecting the inoculum with a syringe in the cylindrical cavity. After inoculation, the plug (upper 5 mm) was reinserted, sealed with sterile paraffin, and covered with a small piece of wet absorbent cotton to prevent dehydration. High humidity (>90%) and 30 to 33°C temperature was maintained after inoculation in a glasshouse. After 4 to 10 days, fruits showed FRD symptoms. The reisolated bacterium from artificially inoculated symptomatic fruits was identical with the original inoculated bacterium. Identity of the bacterial pathogen for FRD was confirmed by phenotypic and genotypic methods. The causal bacterium was a gram-negative, non-sporing motile rod with a single polar flagellum. The bacterium produced yellowish green and blue-green diffusible pigments on King's B (KB) medium. On yeast dextrose calcium carbonate agar at 30°C, the colonies produced abundant, blue, diffusible pigment within 48 h. The bacterium grew at temperatures up to 42°C but not at 4°C. Excellent growth occurred on Salmonella-Shigella agar and MaConkey's medium, as reported also for Pseudomonas aeruginosa strain P8. The bacterium produced ammonia, hydrogen sulfide, arginine dihydrolase, urease, lipase, catalase, gelatinase, and casinase but not amylase, indole, or acetyl methyl carbinol. The bacterium was identified as P. aeruginosa using Biolog based Bacterial Identification System version 4.2 (Biolog Inc., Hayward, CA). The bacterium did not utilize cellobiose, dulcitol, maltose, sorbitol, sucrose, arabinose, and starch. Upon infiltration on tobacco leaves (Nicotiana tabacum cv. Xanthi) at 107 or more cells ml–1, the bacterium gave a strong hypersensitive reaction within 24 h. Transmission electron micrographs (TEM, KYKY 1000B, Japan) of the causal bacterium revealed a single, polar flagellum. Identity was further confirmed as P. aeruginosa based on 16S rRNA sequence (1,491 nt) analysis with universal primers F1 (5′-GAGTTTGATCCTGGCTCAG-3′) and R13 (5′-AGAAAGGAGGTGATCCAGCC-3′). A blastN search of GenBank revealed a >99% nt identity with P. aeruginosa strain TAUC 7 (HQ914782). The 16S rRNA gene sequence (1,491 nt) was deposited in Bankit GenBank (JF797204). To our knowledge, this is the first report of fruit rot of tinda caused by P. aeruginosa in India (ITCC B0030) and a new record of bacterial rot of Praecitrullus fistulosus induced by a fluorescent and blue-green pigment producing P. aeruginosa. To date, P. syringae pv. lachrymans and a nonfluorescent P. pseudoalcaligenes subsp. citrulli were reported to infect Citrullus lanata (1) and Praecitrullus fistulosus (2), respectively. References: (1) D. L. Hopkins and N. C. Schenck. Phytopathology 62:542, 1972. (2) N. W. Schaad et al. Int. J. Syst. Bacteriol. 28:117, 1978.

Update on Huanglongbing Progression and Current Research in Florida

Citrus Huanglongbing (HLB) or citrus greening disease has been in Florida since at least 2005 and has spread to all of the citrus producing regions. The Asian citrus psyllid (Diaphorina citri) is the insect that transmits the suspected causal bacterium which is tentatively named Candidatus Liberibacter asiaticus. Symptoms of the disease are first the yellow shoot (huanglongbing) and the development of the blotchy mottle symptom in the leaves. Mineral deficiency symptoms are often found on infected trees and may mimic normal deficiency symptoms. Fruit on infected trees may be small and lopsided and may abscise prematurely and therefore productivity is reduced. Tree decline eventually occurs. The suspected causal bacterium has not been obtained in pure culture and a polymerase chain reaction (PCR) test along with symptoms, is the only way to verify infected trees. All citrus cultivars appear susceptible. Current management strategies are chemical and biological control to reduced psyllid populations, inspections for infected trees and removal of infected trees to reduce the available pathogen inoculum. Current research on psyllid control, psyllid transmission, symptomology, detection, culturing the causal agent, development of new detection methods, alternative hosts of the psyllid and the causal agent, physiology of the disease, resistant or tolerant cultivar development and more is discussed. HLB is a major problem for citrus production and often limits commercial production. Paper published with permission.

Histopathology of Red Stripe of Rice

A histological study of red stripe of rice was conducted to elucidate the mode of infection of the causal bacterium Microbacterium sp. When pin-point-sized spots first appeared at 3 days after inoculation, the bacterial cells had entered through stomata and multiplied in the intercellular spaces of substomatal parenchymatous tissues. With the early appearance of small yellow spots at 4 to 5 days after inoculation, the bacterium was detected in some xylem vessels as well as in parenchymatous tissues, and it had apparently translocated directly from parenchymatous tissues to transverse vascular systems through spiral vessel walls. With the appearance of typical red stripe symptoms comprised of orange lesions and halos at 8 days after inoculation, bacterial masses were present in transverse and longitudinal vascular bundles in areas with orange lesions. In the areas with orange to light brown spots, granules that stained dark blue using Stoughton's method appeared in the protoplasm of the host parenchymatous cells, which later became necrotic. In halo areas, bacterial masses were observed only in some cases, and chloroplasts were disorganized. Bacterial infection was also confirmed by observing sections of naturally infected samples, and the distribution of bacteria was much more extensive than in artificially inoculated samples.

Serratia marcescens, a Phloem-Colonizing, Squash Bug -Transmitted Bacterium: Causal Agent of Cucurbit Yellow Vine Disease

Cucurbit yellow vine disease (CYVD), which can inflict heavy losses to watermelon, pumpkin, cantaloupe, and squash in U.S. production areas from the midwest to northeastern states, causes phloem discoloration, foliar yellowing, wilting, and plant decline. Bacteria were cultured from the phloem of crown sections of symptomatic plants of Citrullus lanatas and Cucurbita pepo. Those bacteria testing positive in CYVD-specific polymerase chain reaction (PCR) were all gram negative and appeared morphologically identical, producing creamy white, smooth, entire, convex colonies on Luria-Bertani or nutrient agar. Characterized cucurbit-derived strains of Serratia marcescens were introduced into greenhouse-grown squash plants by puncture inoculation and into field-grown squash plants by enclosure with S. marcescens-fed squash bugs, Anasa tristis. Up to 60% of the bacteria-inoculated plants in the greenhouse and up to 17% of field plants caged with inoculative squash bugs developed phloem discoloration and tested positive for S. marcescens by CYVD-specific PCR. None of the controls developed phloem discoloration or tested positive by PCR. Of the diseased field plants, 12% (2 of 35) also yellowed, wilted, and collapsed, exhibiting full symptom development of CYVD. However, neither plant collapse nor decline was observed in the greenhouse-grown, puncture-inoculated plants. The morphology, growth habit, and PCR reaction of bacteria cultured from crown tissue of a subset of plants in each experimental group were indistinguishable from those of the inoculum bacteria. Evidence presented from our studies confirms that the squash bug can transmit S. marcescens, the CYVD causal bacterium. The S. marcescens-A. tristis relationship described here is the first instance in which the squash bug has been identified as a vector of a plant pathogen. Our experiments represent a completion of the steps of Koch's postulates, demonstrating that S. marcescens is the causal agent of CYVD and that the squash bug, A. tristis, is a vector of the pathogen.

Effects of Date of Inoculation on the Within-Plant Movement of Xylella fastidiosa and Persistence of Pierce's Disease Within Field Grapevines

The effects of date of inoculation on the development of Pierce's disease (PD) were evaluated in California grapevines during 1997 through 2000 at four locations. Some vines that had been inoculated either by using blue-green sharpshooters (Graphocephala atropunctata) as vectors or mechanically by needle puncture with the PD causal bacterium Xylella fastidiosa became infected during each month and at each location where infection was attempted. Vines inoculated on the earliest inoculation dates (April to May) developed more extensive and severe PD symptoms, and only 54% of these vines recovered from PD after the following winter, compared with vines that had been inoculated during June through August, of which 88% recovered from PD after the following winter. For the 1999 inoculations, the number of vines infected at a central California site (Parlier) was higher than the number of vines infected at a north coastal site (Hopland). For the best-fitting regression equation, percent recovery of vines infected with X. fastidiosa increased significantly with date of inoculation (r2 = 0.737) at all sites excluding Hopland. The Hopland site had the highest percentage of vines that recovered from PD (100%). At most sites, only early infection (April and May) resulted in chronic disease unless the vines were inoculated at the bases instead of the distal tips of canes. Vines inoculated early in the growing season (April and May) have less chance to recover from Pierce's disease than vines inoculated later (July and August).