Publications

4424

Abstract A new distribution map is provided for Candidatus Liberibacter asiaticus Jagoueix et al. Alphaproteobacteria: Rhizobiales: Rhizobiaceae. Hosts: Citrus spp. Information is given on the geographical distribution in Africa (Egypt, Ethiopia, Kenya, Mauritius, Réunion, Tanzania, Uganda), Asia (Bangladesh, Bhutan, Borneo, Cambodia, China, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Hunan, Jiangxi, Sichuan, Yunnan, Zhejiang, Hong Kong, India, Andhra Pradesh, Arunachal Pradesh, Assam, Bihar, Delhi, Gujarat, Haryana, Himachal Pradesh, Jammu and Kashmir, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Manipur, Meghalaya, Mizoram, Nagaland, Odisha, Punjab, Rajasthan, Sikkim, Tamil Nadu, Tripura, Uttar Pradesh, Uttarakhand, West Bengal, Indonesia, Java, Lesser Sunda Islands, Provinsi Papua, Sulawesi, Sumatra, Iran, Japan, Kyushu, Ryukyu Islands, Laos, Malaysia, Peninsular Malaysia, Sarawak, Myanmar, Nepal, Oman, Pakistan, Philippines, Saudi Arabia, Sri Lanka, Taiwan, Thailand, Vietnam, Yemen), North America (Barbados, Belize, Costa Rica, Cuba, Dominica, Dominican Republic, Guadeloupe, Guatemala, Honduras, Jamaica, Martinique, Mexico, Nicaragua, Panama, Puerto Rico, Trinidad and Tobago, Unites States Virgin Islands, USA, Alabama, California, Florida, Georgia, Louisiana, South Carolina, Texas), Oceania (Papua New Guinea, Timor Leste), South America (Argentina, Brazil, Bahia, Minas Gerais, Paraná, São Paulo, Colombia, Paraguay, Venezuela).

Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid-associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, “Candidatus Profftella armature,” “Candidatus Carsonella ruddii,” CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

Summary Large amounts of detrital organic matter and osmolytes accumulate in the sediments of hadal trenches (>6000 m depth) due to the funnelling effect. It is still unknown whether there are novel active microbes that depend on specific carbon sources in extreme and isolated environments. In this study, we present a novel active bacterial phylum, Candidatus Tianyabacteria in the FCB superphylum, which was enriched in sediments collected from the Challenger Deep. Genome binning resulted in high‐quality Ca . Tianyabacteria genomes representing two Ca . Tianyabacteria lineages (L1 and L2) in sediments 0–21 cm below the surface (cmbsf); L1 tends to be abundant in the upper layers (0–9 cmbsf), and L2 seems to be more prevalent in the deeper layers (12–21 cmbsf). Gene annotation and transcriptomics results indicate that the two lineages might import and catalyse amino acids and myo‐inositol into central carbon metabolism for a heterotrophic lifestyle. Probably due to differences in environmental oxygen levels, the L2 genomes harbour gene clusters responsible for denitrification and fermentation, while the L1 genomes encode octahaem cytochrome c and multicopper oxidase using unknown substrates. The Ca . Tianyabacteria are thus novel heterotrophic organisms that participate in processes of carbon, nitrogen and organic osmolyte cycling in hadal sediments.

‘Candidatus Liberibacter asiaticus’ (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old ‘Valencia’ sweet orange on ‘Kuharske’ citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 109 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 1010 cells/tree, and 6.0 × 1010 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 106 cells/leaf or 9.2 × 104 cells cm–2 in leaves, approximately 104 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.