Draft Genome Sequence of Pseudomonas mediterranea Strain CFBP 5447T, a Producer of Filmable Medium-Chain-Length Polyhydroxyalkanoates.

Pseudomonas mediterranea strain CFBP 5447(T) is a phytopathogenic bacterium isolated from tomato plants affected by pith necrosis disease. Moreover, its ability to produce medium-chain-length polyhydroxyalkanoates (mcl-PHAs) in culture from different carbon sources and valuable microbial products, such as cyclic lipopeptides, has been well documented. Here, we report the first draft genome sequence of this species.

Draft genome sequence of Pseudomonas corrugata, a phytopathogenic bacterium with potential industrial applications.

Pseudomonas corrugata was first described as the causal agent of a tomato disease called 'pith necrosis' yet it is considered as a biological resource in various fields such as biocontrol of plant diseases and production of industrially promising microbial biopolymers (mcl-PHA). Here we report the first draft genome sequence of this species.

Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis-single-strand conformation polymorphisms.

Citrus tristeza virus (CTV) is the causal agent of tristeza disease, which is one of the most devastating diseases of citrus crops worldwide. This paper describes a method for the rapid detection and genotyping of naturally spreading CTV isolates. This method uses ELISA or dot-blot immunological tests to detect trees infected with CTV. The reaction wells or membrane spots for which there is a positive reaction are sequentially treated by (i) washing and elution of viral RNA from the trapped samples, (ii) one-step synthesis of cDNA and PCR and (iii) automated fluorescence-based capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis of amplification products. Comparative CE-SSCP results are presented for CTV RNA extracted directly from infected leaves and ELISA plates or from membranes. In the analyses of all of these RNA samples, the p18, p27 and p23 CTV genes were targeted for amplification. Specific profiles of forward and reverse strands were obtained from a group of eight CTV isolates collected in Sicily, each with distinct biological characteristics, which were analyzed using the conventional two-step procedure (immunological detection followed by CE-SSCP molecular characterization after RNA isolation) or in a continuous process of ELISA/CE-SSCP or dot-blot/CE-SSCP starting from infected plant material. The combined method is simple, highly sensitive and reproducible, thus allowing the processing of numerous field samples for a variety of epidemiological needs. The sequential processing of an ELISA or dot-blot/ELISA followed by CE-SSCP is expected to allow the rapid detection of recent CTV infections along with the simultaneous characterization of the genetic diversity and structure of the population of newly invading CTV.

Detection of Multiple Infections of Citrus exocortis viroid, Citrus viroid III, and Hop stunt viroid Variants in Hunan Province, China.

Citrus cultivation in China has increased since the late 1970s, with China now having the largest area of citrus in culture in the world that is spread in 22 provinces and municipalities. Hunan Province has undergone a program to become one of the major citrus producers in China. Poncirus trifoliata is the main rootstock, so citrus viroids are a limiting factor for further citriculture development. In mainland China, only the presence of Citrus exocortis viroid (CEVd) has been reported from Etrog citron indexing, sPAGE (sequential polyacrylamide gel electrophoresis) analysis (2), and reverse transcription (RT)-PCR (3). Three viroid-like RNAs, a1, b1, and d, based on sPAGE patterns were detected years ago in our laboratory in budsticks received from Sichuan Province. To identify different viroids and determine their distribution, a survey has been undertaken. Field trees showing stunting, bark scaling and cracking of the rootstock, and poor yield were tested using biological indexing and PCR for the most frequent citrus viroids. Samples from six trees of a local sweet orange variety and three of a Clementine variety introduced from abroad, both grafted on P. trifoliata and showing a variable degree of bark scaling and cracking, were collected near Changsha and in the County of Xin Ning at the end of summer 2006. Small pieces of bark were inserted in stems of young E. citron budwood grafted on rough lemon and maintained in a warm greenhouse (24 to 32°C). Indexing on E. citron showed mild epinasty and leaf roll typical of citrus viroid infections. To identify specific viroids, bark was ground to a fine powder with liquid nitrogen and total RNA was extracted with TRIZOL Reagent (Invitrogen, San Diego, CA) and tested by RT-PCR to detect CEVd, Hop Stunt viroid (HSVd), and Citrus viroid III (CVd-III), as well as to identify the cachexia variants of HSVd. Four primer pairs were used to test the RNA extracts by RT-PCR (1). All samples were infected by HSVd, eight with CVd-III, and six with CEVd. The cachexia variants of HSVd were detected in four of nine samples. Mixed infections were as follows: one sample had CEVd and HSVd, eight had HSVd and CVd-III, and five were infected by the three viroids. A second sampling 3 months after inoculation gave the same amplification patterns. The results show that at least three viroids are present in citrus orchards in Hunan Province. To our knowledge, this is the first report of cachexia variants of HSVd and CVd-III in China. The common occurrence of these viroids supports the need for proper indexing of mother trees and a specific shoot tip grafting program to create healthy budwood sources to provide healthy plants. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes, 20:105, 2006. (2) L. Han et al. Viroids. CSIRO Publishing, Melbourne, 283, 2003. (3). Q. Hu et al. Acta Bot. Sin. 39:613, 1997.

Citrus psorosis virus Bark Scaling on Tarocco Sweet Orange.

Despite the wide distribution of vein flecking of citrus leaves in Italy, psorosis bark scaling has been reported only on Navelina sweet orange (Citrus sinensis L.), and Thompson and Washington navel oranges (1). Infection has not been found on any local sweet orange cultivars. Among these is Tarocco, a sweet orange cultivar that originated in Sicily, is very common because of the high fruit quality, has an attractive fruit appearance, and has blood-red pigmented flesh. In April 2001, classic psorosis bark scaling symptoms were observed on the main limbs of 10-year-old Tarocco trees grafted on sour orange rootstock, originally obtained by topworking Clementine (C. reticulata Blanco) trees with scions collected from 19-year-old Tarocco trees with no bark scaling at that time. The symptomatic trees first displayed one or two isolated circular patches of scales with gumming on the main or secondary limbs. As the disease progressed, the number of patches increased and coalesced to form bigger scales, resulting in bark flaking. Approximately 15% of trees in the field showed different stages of the disease. All of the affected trees showed vein flecking of young leaves. A leaf pattern was also present in a few plants without bark symptoms. Bark symptoms were correlated with the presence of Citrus psorosis virus (CPsV). Samples (110 representing 10% of the total number of trees in a field located in the area of Catania, Sicily) were collected during the spring flush using a W-pattern sampling method and tested by double-antibody sandwich enzyme-linked immunoassay (DAS-ELISA) (monoclonal antibody [MAb] PS29) (2). Of trees tested, 14% showed bark scaling, and 86% were symptomless. All symptomatic plants were tested and 70% of symptomless trees were positive based on DAS-ELISA. To confirm DAS-ELISA results, 10 field samples were also tested by bioassay on indicator plants (Navelina sweet orange ISA 315 and Pineapple sweet orange), triple-antibody sandwich enzyme-linked immunoassay (TAS-ELISA), and immunosorbent electron microscopy (ISEM) with a different antiserum (MAb 13C5). DAS-ELISA-positive samples produced vein flecking on indicator plants, were positive based on TAS-ELISA, and contained typical CPsV particles based on ISEM (R. G. Milne, IFA, CNR, Turin). To our knowledge, this is the first demonstration of psorosis bark scaling reaction of Tarocco sweet orange due to CPsV infection. References: (1) A. Catara et al. Proc. Int. Soc. Citri. 1:426, 1981. (2) M. Tessitori et al. Proc. 15th Conf. IOCV, IOCV, Riverside. In press.

Reaction of citrus rootstocks to Meloidogyne javanica.

The response of Citrus spp. and related rootstocks to a population of Meloidogyne javanica was evaluated in a screenhouse experiment. Palestine and Rangpur lime, rough lemon, sour orange, Sexton and Thentriton tangelo, and Volkamer lemon were not infected by M. javanica. Galls and tip swellings were observed on the roots of Poncirus triloliata and Troyer citrange. There was no evidence of nematode development. Symptoms induced by the nematode were stelar division, syncytia formation in the vascular tissues, and necrotic cells.