Exploiting the microbiome associated with normal and abnormal sprouting rice (Oryza sativa L.) seed phenotypes through a metabarcoding approach.

Rice germination and seedlings' growth are crucial stages that influence crop establishment and productivity. These performances depend on several factors, including the abundance and diversity of seed microbial endophytes. Two popular rainfed rice varieties cultivated in Cameroon, NERICA 3 and NERICA 8, were used for investigating the seed-associated microbiome using the Illumina-based 16 S rRNA gene. Significant differences were observed in terms of richness index between normal and abnormal seedlings developed from sprouting seeds, although no significant species evenness index was assessed within either phenotype. Two hundred ninety-two bacterial amplicon sequence variants were identified in seed microbiome of the rice varieties, and principal coordinate analysis revealed that microbial communities formed two distinct clusters in normal and abnormal seedling phenotypes. Overall, 38 bacteria genera were identified, belonging to 6 main phyla. Furthermore, the core microbiome was defined, and the differential abundance of 28 bacteria genera was assessed. Based on the collected results, putative bacterial genera were directly correlated with the development of normal seedlings. For most genera that are recognised to include beneficial species, such as Brevundimonas, Sphingomonas, Exiguobacterium, Luteibacter, Microbacterium and Streptomyces, a significant increase of their relative abundance was found in normal seedlings. Additionally, in abnormal seedlings, we also observed an increased abundance of the genera Kosakonia and Paenibacillus, which might have controversial aspects (beneficial or pathogenic), together with the presence of some genera (Clostridium sensu stricto) that are commonly correlated to sick plants. The putative functional gene annotation revealed the higher abundance of genes related to the metabolic biosynthesis of soluble carbohydrates and starch, tryptophan, nucleotides and ABC transporters in normal seedlings. Data presented in this study may help in further understanding the importance of the seed endophyte microbiome for driving a correct development of rice plants at the early stages and to identify possible beneficial bacteria for technological applications aimed to increase seed quality and crop productivity.

First report of Stemphylium leaf blight of onion (Allium cepa) caused by Stemphylium vesicarium in Italy.

In July 2019, severe leaf symptoms were observed on onion plants (Allium cepa L. cv. Dorata di Parma) in a commercial field located in the municipality of Medicina (Bologna, Emilia-Romagna region), in northern Italy. Diseased leaves showed yellowish-pale-brown and oval-shaped lesions, which later coalesced in larger necrotic areas, and black leaf tips. As the disease progressed, conidia developed on the necrotizing leaves, until premature desiccation of the whole plants. Disease incidence of approximately 70% was calculated in the affected field, together with yield losses that were estimated to be above 30%. Symptomatic tissue fragments excised from the leaf lesions were surface disinfested with NaOCl 1% for 2 minutes, rinsed with sterile water and transferred onto potato dextrose agar (PDA). Fungi were consistently isolated after 5 days of incubation at 27 ± 1°C in the dark. Single spore isolation was performed on PDA to obtain 7 pure cultures, whose morphological characteristics were consistent with the description of Stemphylium vesicarium (Ellis 1971). DNA from a representative single spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers P-ITS1 and P-ITS4 (White et al. 1990). The PCR product was sequenced and deposited in GenBank (Accession No. OP144057). A BLAST search in CBS-KNAW collection bank (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) showed 100% identity for the ITS gene with the strain of S. vesicarium under accession number CBS 124749. Moreover, the PCR assay using the primer pair KES 1999 and KES 2000 (Graf et al. 2016) for the cytochrome b gene displayed the specific fragments of 420 bp for S. vesicarium. The isolate was tested for pathogenicity on onion (potted plants cv. Texas Early Gran, fourth leaf stage) by spraying 4 ml of a conidial suspension (1 × 104 conidia/ ml) per plant. Inoculated and non-inoculated (sprayed with sterile distilled water) plants were kept at 24 ± 1°C and 90% relative humidity with a 16-h photoperiod. Seven days after inoculation, disease assessment was performed. Inoculated plants showed typical Stemphylium leaf blight (SLB) symptoms, similar to those observed in the field. No symptoms developed on the water-inoculated plants. S. vesicarium was consistently reisolated from the artificially inoculated onion plants and identified using a PCR assay, according to Graf et al. (2016). The assay was repeated twice with the same results. SLB is currently reported worldwide and it is considered a re-emerging threat and a truly challenging fungal disease, which can result in yield and quality losses of up to 90% in onion crops (Hay et al. 2021). In Italy, S. vesicarium has been reported several years ago on pear (Ponti et al. 1982) and, more recently, on radish sprouts (Belisario et al. 2008), chili pepper (Vitale et al. 2017) and spinach (Gilardi et al. 2022). To our knowledge, this is the first report of S.vesicarium on onion in Italy. Our results stress that development and implementation of innovative Integrated Pest Management (IPM) strategies are urgently needed to ensure an effective control of SLB, since only a few moderately resistant onion varieties are available (Hay et al. 2021) and no fungicides are currently registered to specifically control SLB in Italy. Further studies are underway to elucidate the pathogen geographic distribution and assess the impact of this disease on the onion crop in Italy.

Plant Growth Promoting and Biocontrol Activity of Streptomyces spp. as Endophytes.

There has been many recent studies on the use of microbial antagonists to control diseases incited by soilborne and airborne plant pathogenic bacteria and fungi, in an attempt to replace existing methods of chemical control and avoid extensive use of fungicides, which often lead to resistance in plant pathogens. In agriculture, plant growth-promoting and biocontrol microorganisms have emerged as safe alternatives to chemical pesticides. Streptomyces spp. and their metabolites may have great potential as excellent agents for controlling various fungal and bacterial phytopathogens. Streptomycetes belong to the rhizosoil microbial communities and are efficient colonizers of plant tissues, from roots to the aerial parts. They are active producers of antibiotics and volatile organic compounds, both in soil and in planta, and this feature is helpful for identifying active antagonists of plant pathogens and can be used in several cropping systems as biocontrol agents. Additionally, their ability to promote plant growth has been demonstrated in a number of crops, thus inspiring the wide application of streptomycetes as biofertilizers to increase plant productivity. The present review highlights Streptomyces spp.-mediated functional traits, such as enhancement of plant growth and biocontrol of phytopathogens.

Gastrointestinal microbial population of turkey (Meleagris gallopavo) affected by hemorrhagic enteritis virus.

Hemorrhagic enteritis (HE) is an acute viral disease that affects avian species, particularly turkeys, compromising their commercial production and having a negative effect on animal welfare. Turkey adenovirus 3 (TAdV-3), is the main causal agent of the disease. In this study, we considered 3 groups of turkeys to achieve 2 purposes: 1) A preliminary investigation on the microbiota content in the 4 parts of healthy turkey's intestine (group A), namely duodenum, jejunum, ileum, and ceca was done; 2) an investigation on the relationship between natural infections with TAdV-3 and the intestinal microbiota in the jejunum, where HE mostly develops, comparing group A with animals with molecular positivity for the virus and with clinical signs of HE (group B) and animals with molecular positivity for the virus but without clinical signs (group C). Massive sequencing of the hypervariable V1-V2 regions of 16S rRNA gene and QIIME 1.9.1 software analysis was performed, and operation taxonomic units (OTUs) were classified into 4 abundant phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The microbial population of small intestine was distributed almost homogeneously in the healthy turkeys, and Firmicutes was the prevalent phylum (79.85% in duodenum, 89.57% in jejunum and 99.28% in ileum). As compared with small intestine, ceca microbial community was much more heterogeneous: Firmicutes (48.03%), Bacteroidetes (33.60%) and Proteobacteria (12.32%). In the natural infections of HEV, the main bacterial families were Bacteroidaceae (Bacteroidetes) and Peptostreptococcaceae (Firmicutes), uniquely detected in group B and C. Also Clostridiaceae (Firmicutes) was detected, uniquely in group B.

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy.

A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

Molecular characterization of Clostridium perfringens strains isolated from diseased turkeys in Italy.

One hundred and six Clostridium perfringens field strains, isolated from diseased turkeys in Italy between 2006 and 2015, were toxinotyped by polymerase chain reaction. Strains were derived from intestines (87), livers (17) and subcutaneous tissues (2). In addition to the four major toxins, strains were also screened for NetB toxin, enterotoxin and beta2 toxin encoding genes. The intestinal gross lesions of turkeys with enteric disorders were statistically studied with respect to the presence of C. perfringens beta2 toxin encoding gene and coccidia in the gut. All the isolates belonged to the toxinotype A and were netB negative. Enterotoxin (cpe) and beta2 toxin (cpb2) encoding genes were detected in two (2.63%) and 76 (71.69%) strains, respectively. Toxinotype results agree with the few published reports concerning the genetic characterization of C. perfringens of turkey origin. On the contrary, the presence of netB and cpb2 genes differs from the results of a previous study where these genes were detected respectively in 6.6% and in 0.5% of the tested strains. Necrotic enteritis in turkeys was not statistically correlated either to the presence of cpb2 gene, or to the synergistic effect operated by coccidia, even though a high percentage of birds with these protozoa in the gut showed necrotic enteritis lesions (64.29%).

Genetic diversity of Escherichia coli isolates of animal and environmental origins from an integrated poultry production chain.

Escherichia coli is a normal inhabitant of the intestinal tract of chickens, but when an imbalance in the gut microbiota occurs, E. coli may overgrow and cause extraintestinal infections. The aims of this study were to assess the distribution and spread of E. coli isolates with specific phylogenetic groups and antimicrobial resistance characters among asymptomatic breeder flocks and their broiler progenies with early symptoms of colibacillosis. Broiler flocks were treated with lincospectin during the first week of life and sampled at one, 21 and 42 days. The majority of the 363 E. coli isolates belonged to phylogenetic group A (53.17%), followed by groups D (23.14%), B1 (19.28%) and B2 (4.41%). In broilers, group A was the most represented in birds of 21 and 42 days of age whereas group B1 was the most represented phylogroup in one-day old chicks. More than 90.00% of the isolates were resistant to one or more antimicrobials. Along the life-time of broilers, no differences were found on the occurrence of resistant isolates except for the number of E. coli with elevated MIC to spectinomycin, which increased significantly after the lincospectin treatment. According to XbaI-macrorestriction analysis, a high genetic diversity among E. coli isolates was underlined. Four antimicrobial resistant E. coli isolates of phylogroups A, B1 and D collected from breeders showed similar PFGE patterns to five isolates collected from the respective broiler progenies suggesting a potential spread of these isolates from breeders to broilers.

Class 1 and class 2 integrons in avian pathogenic Escherichia coli from poultry in Italy.

The aim of this study was to investigate the occurrence of class 1 and 2 integrons in avian pathogenic Escherichia coli (APEC) from poultry in northern Italy. Strains were tested for phenotypic resistance to aminoglycosides and sulphonamides, and the association between the presence of integrons and the resistance to these antimicrobials was evaluated. A total of 299 isolates (158 from turkeys, 110 from broilers, and 31 from layer hens) were collected from 200 industrial farms. Antimicrobial susceptibility test by the disk diffusion method was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. All strains were screened for the presence of class 1 and 2 integrons by PCR and sequencing. About 55% of APEC contained integrons (class 1, 49.8%; class 2, 10.4%). Different variants of the aadA (5 variants) and the dfrA (4 variants) genes, encoding for streptomycin and trimethoprim resistance respectively, were detected in integron-positive isolates. Less common gene cassettes, such as sat, estX, and orfF, were also identified. Fifteen and 4 gene cassette arrays were found among class 1 and 2 integrons, respectively. High levels of resistance were observed for triple sulphonamides (79.3%), streptomycin (67.2%), and sulfamethoxazole combined with trimethoprim (62.2%), whereas resistance against gentamycin (16.7%), kanamycin (14.7%), and apramycin 3.0%) was low. Integron positivity was significantly higher in isolates phenotypically resistant to aminoglycosides (63.6% vs. 37.8%, P<0.001) and sulfonamides (64.1% vs. 21.1%, P<0.001) than in susceptible ones. Integron-borne aminoglycoside and sulfonamide resistance in APEC represents a concern for the poultry industry in Italy, since they are among the most commonly used antimicrobials in poultry therapy.

Continued use of IBV 793B vaccine needs reassessment after its withdrawal led to the genotype's disappearance.

Over a period of almost two years, broilers chickens on several hundred Italian farms, were monitored for infectious bronchitis virus. Detections were genotyped using a hypervariable region of the gene coding for the S1 segment of the spike protein. A range of genotypes were detected which comprised QX, Q1, Mass, D274 and 793B. Sequences of 793B viruses detected in chickens, vaccinated with either of the two commonly used 793B type vaccines were almost identical to sequences of one or other of these vaccines. This strong indication of vaccine association led to the withdrawal of live 793B vaccine use on all of the farms of the study. Except for one sample collected soon after 793B vaccination ceased, it was no longer possible to detect 793B vaccine on these farms. It appears that field 793B strains have disappeared from the region of Italy tested thus obviating any need for current vaccine protection against 793B.

Longitudinal field studies of avian metapneumovirus and turkey hemorrhagic enteritis virus in turkeys suffering from colibacillosis associated mortality.

The aim of this study was to evaluate if the exposure to Avian metapneumovirus (aMPV) and/or to Turkey hemorrhagic enteritis virus (THEV) was significant for the induction of episodes of colibacillosis in aMPV and THEV vaccinated turkeys. Colibacillosis-associated mortality was recorded and longitudinal virological studies performed in three consecutive turkey flocks reared in the same farm. aMPV and THEV diagnostic swabs and blood samples were made once a week up to 14 weeks of age. Swabs were processed by molecular techniques for viruses detection and antibody titres were evaluated. Field subtype B aMPVs were detected in all flocks at different ages of life always associated with respiratory signs and increase of colibacillosis-associated mortality. THEV has been consistently detected in all flocks since the 9th week of age. Vaccination with a single dose of the THEV commercial inactivated vaccine available in Italy seems does not protect the birds from the infection. Sequence comparison of the hexon protein of one of the THEV strains detected, and strains isolated worldwide, revealed high similarity between them. These results are consistent with the notion that the hexon protein, being the major antigenic component of the virus, is highly conserved between the strains. Results showed that field aMPV infection is directly correlated to colibacillosis-associated mortality. Less clear appears the role of THEV because the endemicity of aMPV makes difficult to evaluate its role in predisposing colibacillosis in absence of aMPV. It would be interesting to further investigate this issue through experimental trials in secure isolation conditions.

Rapid detection of subtype B avian metapneumoviruses using RT-PCR restriction endonuclease digestion indicates field circulation of vaccine-derived viruses in older turkeys.

Live vaccines predominantly control avian metapneumovirus (aMPV) infection in poultry flocks, but vaccine virus can be found for extended periods after application. The most frequently used aMPV vaccine in Italy, VCO3 subtype B, was shown to contain a unique Tru9I restriction endonuclease site within the amplicons produced by a commonly used aMPV diagnostic reverse transcriptase (RT)-nested polymerase chain reaction (PCR). Analysis of European and database logged subtype B aMPV sequences confirmed that the sequence occurred only in the VC03 vaccine. A subsequent RT-PCR restriction endonuclease study of field samples, collected from turkeys between 2007 and 2012, detected subtype B vaccine-derived strains in 12 of 90 samples tested that were collected from birds under 12 weeks of age.

Characterization and antimicrobial resistance analysis of avian pathogenic Escherichia coli isolated from Italian turkey flocks.

This study investigated the occurrence of avian pathogenic Escherichia coli (APEC) in a finishing turkey commercial farm, carrying out longitudinal surveys involving 3 consecutive flocks. The diversity and the distribution of the E. coli strains detected during colisepticemia outbreaks were examined. The strains were isolated, serogrouped, assessed for the presence of virulence-associated genes, typed by random amplification of polymorphic DNA (RAPD), and antimicrobial resistance analysis was then carried out. Escherichia coli O78 and O2 were predominantly found. Moreover, based on the somatic antigens used in the study, strains were recovered that were nontypeable. On one occasion, an E. coli O111 strain was found in turkeys. The E. coli isolates differed in terms of antibiotic resistance and RAPD profile. All strains possessed the virulence genes that enabled them to be considered APEC. Strains not only differed between flocks, but also within the same flock. These findings point out the importance of addressing colibacillosis therapy on the basis of a sensitivity test.

Approved medication of water with enrofloxacin to treat turkey colibacillosis: assessment of efficacy using a PK/PD approach.

The efficacy of administering enrofloxacin at 10mg/kg in medicated water to turkeys was evaluated by applying a PK/PD approach to the kinetic parameters obtained after oral pulsed administration and to the MIC values of avian pathogenic Escherichia coli (APEC) strains isolated from commercial turkey flocks. The kinetic parameters of enrofloxacin were evaluated in 10 healthy and 10 colisepticemic turkeys that received the drug dissolved in medicated water at 89 µg/mL and 71 µg/mL, respectively, for 10h/day for 5 days. Blood samples were collected for 24h from all turkeys on the last day of treatment, and the serum was analysed by HPLC with fluorimetric detection. The mean AUC (7374.53±1067.64 h ng/mL and 7656.95±1460.61 h ng/mL) and C(max) values (673.09±186.18 ng/mL and 543.50±68.75 ng/mL) obtained for healthy and sick turkeys were not significantly different. High-level resistance was observed in 30.3% of strains, 40.5% exhibited intermediate resistance, and only 29.2% were susceptible; the MIC(50) and MIC(90) values were 1mg/L and 32 mg/L, respectively. The PK/PD parameters C(max)/MIC(50) (0.67 and 0.54 for healthy and sick turkeys, respectively) and AUC/MIC(50) (7.37 and 7.66) were lower than the efficacy breakpoints reported for fluoroquinolones. These results indicate that authorised dosage of enrofloxacin used in pulsed medicated water administration could be ineffective against more than the 70% of circulating APEC strains, indicating the need to test the drug susceptibility of APEC prior to administering the drug and adopting a more convenient medication schedule for mass treatment.

Phylogenetic analysis of ORF5 and ORF7 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) from PRRS-positive Italian farms: a showcase for PRRSV epidemiology and its consequences on farm management.

We investigated the dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) variability in a range of swine PRRS-positive farms located in Northern Italy, to provide insights into the epidemiology and diffusion of the virus, particularly throughout the entire swine production chain. In this context, we also examined the effectiveness and the critical points of a recently developed gilts acclimatization program in swine breeder farms. To achieve these aims, we designed new primers and determined 64 complete open reading frame 5 (ORF5) sequences, representing Italian PRRSV field strains and the European vaccine Porcilis strain (Intervet); in addition, the more conserved ORF7 of 11 PRRSV strains were sequenced. The domains' prediction of their putative protein sequences was performed as well. Based on these sequences, phylogenetic trees were inferred which revealed a high degree of variability among the PRRSV Italian strains. The outcomes of the phylogenetic analysis showed that the most frequent source of infection in PRRS-positive farms (sow herds, nursery sites, fattening units) was the introduction of animals carrying a new variant and not the modification of already present variants; moreover, the integration of data from phylogenetic analysis and from the clinical and serological status of the swine herds suggested that the acclimatization program could be a valid tool to stabilize the PRRS clinical picture in farms, only when applied in combination with rigorous bio-security routine management and avoid the incoming of new PRRSV variants.