Population structure and diversity of citrus tristeza virus (CTV) isolates in Hunan province, China.

Stem-pitting (SP) is the main type of citrus tristeza virus (CTV) that causes severe damage to citrus trees, especially those of sweet orange, in Hunan province, China. Understanding the local CTV population structure should provide clues for effective mild strain cross-protection (MSCP) of the SP strain of CTV. In this study, markers for the p23 gene, multiple molecular markers (MMMs), and sequence analysis of the three silencing suppressor genes (p20, p23 and p25) were employed to analyze the genetic diversity and genotype composition of the CTV population based on 51 CTV-positive samples collected from 14 citrus orchards scattered around six major citrus-growing areas of Hunan. The results indicated that the CTV population structure was extremely complex and that infection was highly mixed. In total, p23 gene markers resulted in six profiles, and MMMs demonstrated 25 profiles. The severe VT and T3 types appeared to be predominantly associated with SP, while the mild T30 and RB types were related to asymptomatic samples. Based on phylogenetic analysis of the amino acid sequences of p20, p23 and p25, 19 representative CTV samples were classified into seven recently established CTV groups and a potentially novel one. A high level of genetic diversity, as well as potential recombination, was revealed among different CTV isolates. Five pure SP severe and two pure mild strains were identified by genotype composition analysis. Taken together, the results update the genetic diversity of CTV in Hunan with the detection of one possible novel strain, and this information might be applicable for the selection of appropriate mild CTV strains for controlling citrus SP disease through cross-protection.

Deep sequencing and analysis of small RNAs in sweet orange grafted on sour orange infected with two citrus tristeza virus isolates prevalent in Sicily.

Two representative isolates of a citrus tristeza virus population in Sicily, SG29 (aggressive) and Bau282 (mild), were sequenced via viral small RNAs (vsRNA) produced in budlings of sweet orange grafted on sour orange. Phylogenetic relationships with Mediterranean and exotic isolates revealed that SG29 clustered within the "VT-Asian" subtype, whereas Bau282 belonged to the cluster T30. The study confirms that molecular data need to be integrated with bio-indexing in order to obtain adequate information for risk assessment.

Soil Microbial Communities in Lemon Orchards Affected by Citrus Mal Secco Disease.

Mal secco is a vascular disease of citrus caused by the mitosporic fungus Plenodomus tracheiphilus. Soil containing infected plant material constitutes an inoculum source for root infections. In this study, the soil bacterial and fungal communities of five lemon orchards located in Syracuse Province (Sicily, Italy) affected by mal secco were analyzed. Soil samples were collected under lemon tree canopies and subjected to total genomic DNA extraction. The fungal DNA was detected through qPCR in all orchards, with variable concentrations. Bacterial and fungal communities were profiled using 16S and ITS amplicon-based high-throughput sequencing, respectively. According to our results, the relative abundances of the most represented bacterial phyla (e.g., Proteobacteria, Actinobacteriota, Acidobacteriota) changed across the orchards, while in the fungal community, the phylum Ascomycota was dominant, with Basidiomycota and Mortierellomycota abundances fluctuating. On the whole, ß diversity analysis showed significant variation in the composition of the soil microbial communities across the orchards. This result was confirmed by the analysis of the core community (taxa present at ≥ 75% of total samples), where putative beneficial bacteria resulted in significantly enriched fungus-infected soil samples, suggesting complex microbial interactions. Our findings shed light on the composition and diversity of the soil microbiome in lemon orchards with the occurrence of mal secco infections.

Minor Variants of Orf1a, p33, and p23 Genes of VT Strain Citrus Tristeza Virus Isolates Show Symptomless Reactions on Sour Orange and Prevent Superinfection of Severe VT Isolates.

The control of tristeza quick decline (QD) of citrus is based on the use of rootstocks that are tolerant or resistant to the Citrus tristeza virus (CTV), but some of them show bio-agronomic limits. The application of cross-protection (CP) has been insufficiently explored. The present study examined the possibility of QD control by cross-protection (CP) following reports showing the dependence of the CP strategy on the close genetic relationships between the protective and challenging CTV isolates. Taking advantage of deep sequencing technologies, we located six naturally infected trees harboring no-seedling yellow (no-SY) and no QD decline (mild) VT isolates and used these for challenge inoculation with three QD VT isolates. Symptom monitoring showed that all six Sicilian mild no-SY isolates, based on their genomic relatedness and mild symptoms reactions, provide effective protection against the three severe local VT isolates. The differences between the six mild and three severe isolates were confined to just a few nucleotide variations conserved in eight positions of three CTV genes (p23, p33, and Orf1a). These results confirm that the superinfection exclusion (SIE mechanism) depends on close genetic relatedness between the protective and challenging severe VT strain isolates. Ten years of investigation suggest that CP could turn into an efficient strategy to contain CTV QD infections of sweet orange trees on SO rootstock.

Phenotyping Biological Properties of CTV Isolates.

The protocol described is intended to be used alongside molecular methods in order to reveal the relationship between the genome sequence and the biological properties of a single isolate of Citrus tristeza virus complex (CTV). It enables the phenotypic profile of the isolates to be defined and to infer the associated tristeza diseases (decline, seedling yellows, or stem pitting), to assess their aggressiveness or potential cross protectiveness (if any), and to monitor their movement into the host plants and the transmissibility by aphids.

Genotyping Citrus tristeza virus Isolates by Sequential Multiplex RT-PCR and Microarray Hybridization in a Lab-on-Chip Device.

Citrus tristeza virus (CTV) is the largest known plant RNA virus (ca. 20 Kb), with a plethora of isolates conventionally categorized into six main genotypic groups (T36, VT, T3, RB, T68, T30). Each group includes many isolates with different phenotype profiles. Several techniques and protocols, mostly based on RT-PCR analysis of different regions of specific genes, have been developed for managing the diseases caused by CTV. However, more accurate genomic information would help to plan a correct strategy. This chapter describes a pilot protocol based on a sequential multiplex RT-PCR reaction and microarray hybridization in a miniaturized silicon lab-on-chip (LoC) device. The system comprises a set of 12 primers and 44 probes (× 2 replicates), designed on variable genomic regions of 6 genes: 5'UTR, ORF1a, ORF1b (RdRp), p33, p20, and p23. The system can rapidly analyze any genotype diversity associated with field isolates and distinguish the endemic from the non-endemic isolates. The identification of CTV strains is based on a number of probe hybridizations, which varies according to the genotypes present in the isolates and the differences among the genotypes.

Production of Polyhydroxyalkanoates and Extracellular Products Using Pseudomonas Corrugata and P. Mediterranea: A Review.

Some strains of Pseudomonas corrugata (Pco) and P. mediterranea (Pme) efficiently synthesize medium-chain-length polyhydroxyalkanoates elastomers (mcl-PHA) and extracellular products on related and unrelated carbon sources. Yield and composition are dependent on the strain, carbon source, fermentation process, and any additives. Selected Pco strains produce amorphous and sticky mcl-PHA, whereas strains of Pme produce, on high grade and partially refined biodiesel glycerol, a distinctive filmable PHA, very different from the conventional microbial mcl-PHA, suitable for making blends with polylactide acid. However, the yields still need to be improved and production costs lowered. An integrated process has been developed to recover intracellular mcl-PHA and extracellular bioactive molecules. Transcriptional regulation studies during PHA production contribute to understanding the metabolic potential of Pco and Pme strains. Data available suggest that pha biosynthesis genes and their regulations will be helpful to develop new, integrated strategies for cost-effective production.

Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III.

Citrus are natural hosts of several viroids, which are plant pathogens composed exclusively of a non-protein-coding, small single-stranded circular RNA that is able to replicate autonomously in susceptible hosts. They are responsible for symptoms such as stunting, leaf epinasty, and chlorosis. Citrus viroid III (CVd-III) has been long regarded as a possible dwarfing agent of citrus grafted on trifoliate orange and its hybrids. To investigate molecular mechanisms involved in pathogenesis, the messenger RNA (mRNA) differential display technique was here applied to identify genes whose transcription was significantly altered in leaves of Etrog citron (Citrus medica) infected by CVd-III (variant b). Of eighteen genes identified, thirteen were up-regulated by viroid infection, while five were down-regulated. Except for two genes that encode proteins of unknown function, the remaining genes are mainly involved in plant defence/stress responses, signal transduction, amino acid transport, and cell wall structure. Among the up-regulated genes, it is noteworthy a suppressor of RNA silencing that might be involved in viroid and virus pathogenicity. The functions of these genes are discussed.

Pest categorisation of 'Blight and blight-like' diseases of citrus.

The EFSA Panel on Plant Health performed a pest categorisation of 'Blight and blight-like' for the EU territory. Blight is a major disease of citrus. Similar 'blight-like' diseases are also known (e.g. declinio, declinamiento) and are addressed simultaneously with Blight in the present categorisation. The causal agent(s) remain(s) unknown and the potential role of a recently identified citrus endogenous pararetrovirus (Citrus Blight-associated pararetrovirus, CBaPRV) remains to be established. Transmissibility and ability to produce consistent (although poorly specific) symptoms have been demonstrated and a combination of indirect approaches is used, with limits, for diagnosis. There are large uncertainties on the biology of the causal agent(s) and on the epidemiology of the disease, including the transmission mechanism(s) responsible for the observed field spread. Blight has been reported from North, Central and South America, Africa and Oceania but is not known to occur in the EU. It is listed in Annex IIA of Directive 2000/29EC. It has the potential to enter, establish and spread in the EU territory. The main entry pathway (citrus plants for planting) is closed by existing legislation and entry is only possible on minor pathways (such as illegal import). Blight is a severe disease and a negative impact is expected should it be introduced in the EU, but the magnitude of this negative impact is very difficult to estimate. 'Blight and blight like' satisfies all criteria evaluated by EFSA to qualify as a Union quarantine pest. It does not meet the criterion of being present in the EU to qualify as a Union regulated non-quarantine pest (RNQP). Since the identity of the causal agent(s) of the Blight and blight-like disease(s) and the existence and efficiency of natural spread mechanism(s) remain unknown, large uncertainties affect all aspects of the present pest categorisation.

Transcriptome analysis of Pseudomonas mediterranea and P. corrugata plant pathogens during accumulation of medium-chain-length PHAs by glycerol bioconversion.

Pseudomonas corrugata and P. mediterranea are soil inhabitant bacteria, generally living as endophytes on symptomless plants and bare soil, but also capable of causing plant diseases. They share a similar genome size and a high proteome similarity. P. corrugata produces many biomolecules which play an important role in bacterial cell survival and fitness. Both species produce different medium-chain-length PHAs (mcl-PHAs) from the bioconversion of glycerol to a transparent film in P. mediterranea and a sticky elastomer in P. corrugata. In this work, using RNA-seq we investigated the transcriptional profiles of both bacteria at the early stationary growth phase with glycerol as the carbon source. Quantitative analysis of P. mediterranea transcripts versus P. corrugata revealed that 1756 genes were differentially expressed. A total of 175 genes were significantly upregulated in P. mediterranea, while 217 were downregulated. The largest group of upregulated genes was related to transport systems and stress response, energy and central metabolism, and carbon metabolism. Expression levels of most genes coding for enzymes related to PHA biosynthesis and central metabolic pathways showed no differences or only slight variations in pyruvate metabolism. The most relevant result was the significantly increased expression in P. mediterranea of genes involved in alginate production, an important exopolysaccharide, which in other Pseudomonas spp. plays a key role as a virulence factor or in stress tolerance and shows many industrial applications. In conclusion, the results provide useful information on the co-production of mcl-PHAs and alginate from glycerol as carbon source by P. mediterranea in the design of new strategies of genetic regulation to improve the yield of bioproducts or bacterial fitness.

Pest categorisation of Citrus tristeza virus (non-European isolates).

The Panel on Plant Health performed a pest categorisation of non-European isolates of Citrus tristeza virus (CTV) for the EU territory. CTV is a well characterised virus for which efficient detection assays are available. It is transmitted by vegetative multiplication of infected hosts and by aphid vectors. The most efficient one, Toxoptera citricida, has limited EU presence but another one, Aphis gossypii, is broadly distributed. CTV is reported from a range of countries outside the EU and EU isolates are present in seven of the eight citrus-growing member states. Non-EU isolates are not known to occur in the EU and therefore do not meet one of the criteria for being a Union regulated non-quarantine pest. The natural host range of CTV is restricted to Citrus, Fortunella and Poncirus species. CTV non-EU isolates are listed in Annex IIAI of Directive 2000/29/EC and the main pathway for entry, plants for planting, is closed by the existing legislation. CTV isolates may therefore only enter through minor alternative pathways. They have the potential to subsequently spread through plants for planting and through the action of aphid vectors. CTV non-EU isolates are able to cause severe symptoms on a range of citrus crops that EU isolates do not induce. Overall, non-EU CTV isolates meet all the criteria evaluated by EFSA to qualify as Union quarantine pests. The main knowledge gaps and uncertainties concern (1) the status of Rutaceae species other than Citrus, Fortunella and Poncirus as natural hosts for CTV; (2) the potential undetected presence of non-EU CTV isolates in the EU and in particular the prevalence and biological properties of CTV isolates that may be present in ornamental citrus; and (3) the inability of EU CTV isolates apparently related to non-European stem pitting (SP) isolates to cause SP in sweet orange.

Pest categorisation of Satsuma dwarf virus.

The EFSA Panel on Plant Health performed a pest categorisation of Satsuma dwarf virus (SDV) for the EU territory. SDV is a well-known pathogen and the type species of the genus Sadwavirus in the family Secoviridae. SDV is now considered to include several other formerly distinct viruses which are therefore also covered in the present opinion. Citrus species and their relatives represent the main hosts of SDV and efficient diagnostic techniques are available. SDV is listed on some of its known hosts in Annex IIAI of Directive 2000/29/EC. It is transmitted by vegetative propagation of infected hosts and presumably through the soil, but the precise mechanism or vector(s) are still unknown. SDV is present in Asia and is not known to occur in the EU. Therefore, it does not meet this criterion to qualify as a Union regulated non-quarantine pest (RNPQ). Plants for planting represent the main pathway for the entry, but this pathway is closed by existing legislation for the main hosts (Citrus, Fortunella and Poncirus). SDV is, however, able to enter the EU on plants for plants of its unregulated rutaceous or non-rutaceous hosts. Should it be introduced, SDV has the potential to establish and subsequently spread with plants for planting and, possibly, through its poorly characterised natural spread mechanism(s). SDV is able to cause severe symptoms, quality and yield losses on a range of citrus crops. Overall, SDV meets all the criteria evaluated by EFSA to qualify as a Union quarantine pest. The main knowledge gaps and uncertainties concern (1) the potential significance of the unregulated rutaceous and non-rutaceous hosts for virus dissemination and epidemiology, (2) the origin and trade volume of the plants for planting of these host imported in the EU and (3) the efficiency of natural spread of SDV under EU conditions.

Pest categorisation of Tatter leaf virus.

The EFSA Panel on Plant Health performed a pest categorisation of Citrus tatter leaf virus (CTLV) for the EU territory. This virus is the causal agent of tatter leaf and graft incompatibility in trifoliate orange (Poncirus trifoliata) and its hybrids. CTLV is now recognised as a synonym of Apple stem grooving virus (ASGV), the type Capillovirus species, for which efficient diagnostics are available. There are no known ASGV vectors. The virus is reported in citrus from many countries. In the EU, while ASGV is widely present on apple and pear, it has never been reported on citrus. Since the citrus plants for planting pathway is closed by existing legislation, the main pathway for entry is plants for planting of other host species. In the EU, the high prevalence of ASGV in non-citrus hosts, but its absence in citrus ones suggests that interspecific host transfers are rare. However, there are high uncertainties on the importance and specifics of such host change events. No limits to the establishment of ASGV are identified and spread is likely through the vegetative propagation and trade of infected hosts. Infection of sensitive citrus rootstocks leads to stunted growth and decline of the entire plant a few years after grafting. The rootstocks that are now widely used to prevent citrus tristeza decline are the most affected. Among the criteria evaluated by EFSA for an organism to qualify as a Union quarantine pest, ASGV does not meet the criterion of being absent from or under official control in the EU territory. ASGV satisfies all the criteria evaluated by EFSA to qualify as a Union regulated non-quarantine pest. The main uncertainties concern the possible unreported presence of ASGV in citrus in the EU, the existence and efficiency of interspecific host transfers and the existence of ASGV natural spread.

Pest categorisation of naturally-spreading psorosis.

The EFSA Panel on Plant Health performed a pest categorisation of naturally-spreading psorosis of citrus for the European Union. Naturally-spreading psorosis is poorly defined, because the status of both the disease and its causal agent(s) is uncertain. However, Citrus psorosis virus (CPsV) is a well- characterised Ophiovirus that is systematically associated with the psorosis disease and therefore considered to be its causal agent. Efficient diagnostics are available for CPsV. It is present in at least three EU MS. Naturally-spreading psorosis is currently regulated by Directive 2000/29/EC, while CPsV is not explicitly mentioned in this Directive. CPsV has the potential to enter, establish and spread in the EU territory. However, the main pathway for entry is closed by the existing legislation so that entry is only possible through minor alternative pathways. Plants for planting are the major means of spread while there are uncertainties on the existence and efficiency of a natural spread mechanism. CPsV introduction and spread in the EU would have negative consequences on the EU citrus industry. Of the criteria evaluated by EFSA to qualify as a Union quarantine pest or as a Union regulated non-quarantine pest (RNQP), Naturally-spreading psorosis does not meet the criterion of being a well characterised pest or disease. As it is not explicitly mentioned in the legislation, it is unclear whether CPsV meets the criterion of being currently regulated or under official control. It meets, however, all the RNQP criteria. The key uncertainties of this categorisation concern: (1) the causal role of CPsV in the psorosis disease as well as elements of its biology and epidemiology, (2) the exact nature of the Naturally-spreading psorosis syndrome and the identity of its causal agent and, consequently, (3) whether CPsV should be considered as being covered by the current legislation.

Pest categorisation of Citrus leprosis viruses.

The EFSA Panel on Plant Health performed a pest categorisation of the Citrus leprosis viruses for the EU territory and identified five distinct viruses, Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), Hibiscus green spot virus 2 (HGSV-2), the Citrus strain of Orchid fleck virus (OFV) and Citrus leprosis virus N sensu novo (CiLV-N) as causing this severe disease, most significantly in sweet orange and mandarin. These viruses have in common that they do not cause systemic infections in their hosts and that they all are transmitted by Brevipalpus spp. mites (likely but not confirmed for HGSV-2). Mites represent the most important means of virus spread, while plants for planting of Citrus are only considered of minor significance. These well characterised viruses occur in South and Central America. Leprosis is currently regulated in directive 2000/29 EC and, together with its associated viruses, has never been recorded in the EU. All five viruses have the potential to enter into, establish in and spread within the EU territory, with plants for planting of non-regulated hosts, fruits of Citrus and hitch-hiking of viruliferous mites identified as the most significant pathways. Given the severity of the leprosis disease, the introduction and spread of the various viruses would have negative consequences on the EU citrus industry, the magnitude of which is difficult to evaluate given the uncertainties affecting the Brevipalpus spp. vectors (identity, distribution, density, transmission specificity and efficiency). Overall, leprosis and its five associated viruses meet all the criteria evaluated by EFSA to qualify as Union quarantine pests, but do not fulfil those of being present in the EU or of plants for planting being the main spread mechanism to qualify as Union regulated non-quarantine pests. The main uncertainties affecting this categorisation concern the Brevipalpus spp. mite vectors.

Production of filmable medium-chain-length polyhydroxyalkanoates produced from glycerol by Pseudomonas mediterranea.

Glycerol is an effective carbon source for the production of scl- and mcl-polyhydroxyalkanoates (PHAs) by Pseudomonas spp. P. mediterranea 9.1 (CFBP 5447) synthesizes an amorphous mcl-PHA when grown on crude glycerol, whereas on both reagent grade (RG) and partially refined (PR) glycerol, it produces two very similar distinctive mcl-PHAs with the unusual property of producing, with the appropriate treatment, a transparent film. Mcl-PHAs recovered after biomass extraction have an average molecular weight of approximately 56,000/63,000 Da. The monomer composition and physicochemical properties of such mcl-PHAs suggest their potential application as a softener of biopolymeric blends for food packaging and medical devices.

Emergence and phylodynamics of Citrus tristeza virus in Sicily, Italy.

Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and re-circulation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.

Microarray analysis of Etrog citron (Citrus medica L.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection.

Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants.

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.

Regulation of polyhydroxyalkanoate synthases (phaC1 and phaC2) gene expression in Pseudomonas corrugata.

In this study we examined polyhydroxyalkanoate (PHA) synthases phaC1 and phaC2 gene expression in two strains of Pseudomonas corrugata (Pc) grown in a minimum mineral medium with related (oleic acid and octanoate) or unrelated (glucose) carbon sources. Analysis of transcription was performed by Northern blot and conventional reverse transcriptase (RT) polymerase chain reaction (PCR). In addition, we developed a RT-real-time PCR method to quantitatively evaluate phaC1 (Pc) and phaC2 (Pc) gene expression. Primers and a TaqMan probe were designed for the specific detection of both synthase transcripts as well as of the housekeeping 16S rRNA, and the relative expression of target genes was calculated. We showed that phaC1 (Pc) and phaC2 (Pc) were not cotranscribed and, on the contrary, were independently regulated. In cultures grown with oleic acid as the sole carbon source, only the expression of phaC1 (Pc) was induced (a tenfold increase after 72 h of culture), whereas that of phaC2 (Pc) remained unchanged. In cultures grown with glucose or sodium octanoate, the expression of both phaC1 (Pc) and phaC2 (Pc) was upregulated but at different rates. Cellular PHA content was compared to the gene expression of the PHA synthases and significant correlations were found between PHA production and phaC1 (Pc)/phaC2 (Pc) expression.