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.

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.

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.

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.

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.