High clonality of Mycobacterium avium subsp. paratuberculosis field isolates from red deer revealed by two different methodological approaches of comparative genomic analysis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the aetiological agent of paratuberculosis (Johne's disease) in both domestic and wild ruminants. In the present study, using a whole-genome sequence (WGS) approach, we investigated the genetic diversity of 15 Mycobacterium avium field strains isolated in the last 10 years from red deer inhabiting the Stelvio National Park and affected by paratuberculosis. Combining de novo assembly and a reference-based method, followed by a pangenome analysis, we highlight a very close relationship among 13 MAP field isolates, suggesting that a single infecting event occurred in this population. Moreover, two isolates have been classified as Mycobacterium avium subsp. hominissuis, distinct from the other MAPs under comparison but close to each other. This is the first time that this subspecies has been found in Italy in samples without evident epidemiological correlations, having been isolated in two different locations of the Stelvio National Park and in different years. Our study highlights the importance of a multidisciplinary approach incorporating molecular epidemiology and ecology into traditional infectious disease knowledge in order to investigate the nature of infectious disease in wildlife populations.

Thermoregulatory responses and ingestive behavior of sheep subjected to water restriction and high- and low-energy diets in a semi-arid environment.

The aim of the study was to evaluate the effect of water restriction and low- and high-energy diets on sheep's thermoregulatory responses and ingestive behavior. Forty sheep, non-castrated, with an average body weight of 18.85 kg (SD = 2.80 kg) and an average age of 5 months were assigned to a 2 × 2 factorial arrangement, comprising 2 diets (high- and low-energy) and 2 water offers (ad libitum and 50% water restriction), with 10 replicates. Thermoregulatory responses were evaluated in two periods (morning and afternoon). There was an interaction effect of Diet x Water supply x Periods on respiratory rate (P < 0.05). High-energy diets resulted in increased heart rate, idleness, dry matter feeding and rumination efficiency, and water intake. Low-energy diets increased feeding time, rumination time, the number of ruminal cuds, chews per day, total chewing time, neutral detergent fiber intake and rumination efficiency, number of ruminations per day, average duration of rumination, and defecation frequency. Water supply affected heart rate and idleness (P < 0.05). Sheep had higher values of heart rate and rectal and surface temperatures during the afternoon (P < 0.05). Water restriction combined with a low-energy diet and high environmental temperature leads to a reduction in the respiratory rate of Santa Inês crossbred sheep. Regardless of the dietary energy value, water restriction by 50% of the daily requirement of sheep reduces dry matter intake and increases idleness.

A roadmap of tissue culture and biotechnology in European hazelnut (Corylus avellana L.).

The increasing interest in European hazelnut (Corylus avellana L.) cultivation registered in the last years has led to a significant increase in worldwide hazelnut growing areas, also involving regions characterized by a marginal presence of hazelnut orchards. Despite this increasement, world production still relies on the cultivation of few varieties, most of which are particularly suitable to the environment where they have been selected. Therefore, it is necessary to develop new cultivars with high environmental plasticity capable of providing constant and high-quality productions in the new environments and under the climatic change conditions of traditional growing areas. Over the years, many molecular markers for genetic breeding programs have been developed and omics sciences also provided further information about the genetics of this species. These data could be of support to the application of new plant breeding techniques (NPBTs), which would allow the development of cultivars with the desired characteristics in a shorter time than traditional techniques. However, the application of these methodologies is subordinated to the development of effective regeneration protocols which, to date, have been set up exclusively for seed-derived explants. A further aspect to be exploited is represented by the possibility of cultivating hazelnut cells and tissues in vitro to produce secondary metabolites of therapeutic interest. This review aims to consolidate the state of the art on biotechnologies and in vitro culture techniques applied on this species, also describing the various studies that over time allowed the identification of genomic regions that control traits of interest.

Non-invasive methods to quantify the carcass parameters of sheep: Interaction between thermal environment and residual feed intake.

The thermal environment is important in unit production because the perception of thermal stress can reduce fertility, and productive performance, therefore its management is necessary. The use of non-invasive methods, such as infrared thermography and real-time ultrasonography, are widely used to evaluate indicators in animal production, without the need to slaughter the animals. Thus, we aimed to assess the effect of the thermal environment on the physiological parameters and carcass characteristics of Dorper sheep with positive and negative residual feed intake (RFI) using infrared thermography and real-time ultrasonography techniques. Twenty uncastrated male Dorper sheep (17.8 ± 2.4 kg) were confined for 40 days for RFI classification. Sheep were separated into positive RFI (n = 10) and negative RFI (n = 10). The experimental design was in randomized blocks, in a 2 × 2 factorial arrangement, with 2 thermal environments (full sun or shade) and two feed efficiency groups (positive RFI or negative RFI), with 5 replications. The sheep remained in confinement for 60 days. The animals were slaughtered at the end of the experiment and the carcasses dissected for tissue separation. Rectal temperature (RT) and respiratory rate (RR) were measured at two times (14:00 h and 18:00 h) for periods of 5 days. The RR was determined by indirect auscultation of heart sounds at the level of the laryngotracheal region. The RT was measured introduced a digital clinical thermometer into the animal's rectum. Surface temperature (ST) was obtained using a thermographic infrared camera, collecting the temperatures of the eyeball and skin surface in the regions of the head, ribs, rump, flank and shin. Sheep confined in full sun showed higher RR (P = 0.0001), ST ribs (P = 0.0020), ST rumb (P = 0.0055), ST flank (P = 0.0001) and heat tolerance coefficient (HTC) (P = 0.0010). For sheep confined in full sun, a strong correlation was observed between the RR and the mean ST (MST; r = 0.6826; P = 0.0236) and between the final loin eye area (LEAf) with the real LEA (LEAr) (r = 0.9263; P = 0.0001) and slaughter body weight (SBW) (r = 0.7532; P = 0.0325). For negative RFI sheep, a positive correlation was observed between the RR and the ST rump (r = 0.7343; P = 0.0025) and ST ribs (r = 0.6560; P = 0.0178) and the MST (r = 0.7435; P = 0.0001), between the MST and the LEAr (r = 0.6837; P = 0.0025) and the final LEA (r = 0.6771; P = 0.0144), and between the final LEA and LEAr (r = 0.9942; P = 0.0001), BW (r = 0.8415; P = 0.0277) and MST (r = 0.6771; P = 0.0045). Positive RFI sheep confined to shade showed a high correlation between final LEA and LEAr (r = 0.9372; P = 0.0001). The use of shading in confined Dorper sheep, regardless of the RFI classification, reduces the effects of heat stress on physiological parameters.

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Hybrid de novo genome assembly and comparative genomics of three different isolates of Gnomoniopsis castaneae.

The first genome assemblies of Gnomoniopsis castaneae (syn. G. smithogilvyi), the causal agent of chestnut brown rot of kernels, shoot blight and cankers, are provided here. Specifically, the complete genome of the Italian ex-type MUT401 isolate was compared to the draft genome of a second Italian isolate (GN01) and to the ICMP 14040 isolate from New Zealand. The three genome sequences were obtained through a hybrid assembly using both short Illumina reads and long Nanopore reads, their coding sequences were annotated and compared with each other and with other Diaporthales. The information offered by the genome assembly of the three isolates represents the base of data for further application related to -omics strategies of the fungus and to develop markers for population studies at a local and global scale.

Equus caballus Papillomavirus Type-9 (EcPV9): First Detection in Asymptomatic Italian Horses.

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.

Detection of Equus Caballus Papillomavirus Type-2 in Asymptomatic Italian Horses.

Equine Papillomavirus 2 (EcPV2) is responsible for squamous cell carcinomas (eSCCs) of external genitalia of both male and female horses. However, few studies report the EcPV2 prevalence among healthy horses. Currently, the lack of these data does not permit identifying at-risk populations and, thus, developing screening protocols aimed at the early detection of the infection, as for humans. The aim of our study was to estimate the genoprevalence of EcPV2 in clinically healthy horses in Italy and to evaluate their innate immune response. For this purpose, penile and vulvar swabs of 234 healthy horses were collected through sampling with sterile cytobrushes. Nucleic acids were isolated and EcPV2-L1 presence (DNA) and gene expression (RNA) were checked by RT-qPCR. Our results showed EcPV2-L1 DNA presence in 30.3% of the samples and L1 expression in 48% of the positive samples. No statistically significant differences were found in genoprevalence in relation to sex, age, and origin, while, concerning breeds, the Thoroughbred had the highest risk of infection. Concerning specifically the mares, 40.2% of them resulted in being positive for EcPV2; our findings show a major positivity in pluriparous (p = 0.0111) and mares subjected to natural reproduction (p = 0.0037). Moreover, samples expressing L1 showed an increased expression of IL1B (p = 0.0139) and IL12p40 (p = 0.0133) and a decreased expression of RANKL (p = 0.0229) and TGFB (p = 0.0177). This finding suggests the presence of an effective immune response, which could explain the low incidence of SCCs in positive horses, despite a high EcPV2 genoprevalence (30%).

Retracted: Economic viability in free-range chicken production.

Despite the lack of large-scale farming of free-range chickens in Brazil, their production generates income in the countryside and prevents exodus of rural families in agricultural regions. The objective of this study is to evaluate the economic viability of free-range broiler production in different facilities. The experiment was conducted in two different sheds (masonry shed-SM and wooden shed-SW) located in the Plural Space of the Universidade Federal do Vale São Francisco, municipality of Juazeiro, BA. Here, 200 heavy red French free-range chickens were distributed in the two sheds and were raised from the 1st to the 88th day (slaughter). Assuming that the minimum age for slaughter is 85 days, the results indicated that at least 205 birds in SM and 217 birds in SW were necessary for the producer to earn the minimum per capita monthly wage in Brazil (2020); at least 411 birds in CG and 600 birds in GM were found to be necessary to achieve maximum productivity at the end of the production cycle. The maximum profitability in the slaughter of the chickens was achieved at an age of 60 days.

Less is more: natural variation disrupting a miR172 gene at the di locus underlies the recessive double-flower trait in peach (P. persica L. Batsch).

BACKGROUND: With the domestication of ornamental plants, artificial selective pressure favored the propagation of mutations affecting flower shape, and double-flower varieties are now readily available for many species. In peach two distinct loci control the double-flower phenotype: the dominant Di2 locus, regulated by the deletion of the binding site for miR172 in the euAP2 PETALOSA gene Prupe.6G242400, and the recessive di locus, of which the underlying factor is still unknown. RESULTS: Based on its genomic location a candidate gene approach was used to identify genetic variants in a diverse panel of ornamental peach accessions and uncovered three independent mutations in Prupe.2G237700, the gene encoding the transcript for microRNA miR172d: a ~5.0 Kb LTR transposable element and a ~1.2 Kb insertion both positioned upstream of the sequence encoding the pre-miR172d within the transcribed region of Prupe.2G237700, and a ~9.5 Kb deletion encompassing the whole gene sequence. qRT-PCR analysis confirmed that expression of pre-miR172d was abolished in di/di genotypes homozygous for the three variants. CONCLUSIONS: Collectively, PETALOSA and the mutations in micro-RNA miR172d identified in this work provide a comprehensive collection of the genetic determinants at the base of the double-flower trait in the peach germplasms.

Retraction to: Economic viability in free-range chicken production.

[This retracts the article DOI: 10.1093/tas/txab109.].

Complete genome assembly of the levan-positive strain PVFi1 of Pseudomonas savastanoi pv. savastanoi isolated from olive knots in Central Italy.

Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, is a fluorescent Gram-negative bacterium classified, according to the specific LOPAT profile, as Ib. However, during the 90s, a number of atypical non-fluorescent levan-positive strains of Pseudomonas savastanoi pv. savastanoi have been unexpectedly isolated from olive knots in Central Italy. Since its first report, several studies were conducted on this species variant, but its genome sequence has never been reported. The complete genome sequence and two additional plasmids of PVFi1, a representative strain, were here obtained using a hybrid sequencing approach with both Oxford Nanopore Technology and Illumina sequencing. A thorough genomic analysis unravelled several genetic features of this peculiar strain, showing a transposase insertion downstream a fragmented copy of the levansucrase gene. The same features were previously reported on levan-negative Pseudomonas savastanoi pv. savastanoi strains. In addition, a second copy of the levansucrase gene fully equipped for a gene expression and comparable to the levan-positive Pseudomonas savastanoi pv. glycinea, may explain the levan-positive test. This result provides a solid genetic demonstration that the bacterial species Pseudomonas savastanoi contains either levan-positive or levan-negative strains, providing insights for an update of the related LOPAT classification.

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a ∼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal.

Water intake and ingestive behavior of sheep fed diets based on silages of cactus pear and tropical forages.

The objective was to evaluate the water intake and ingestive behavior of sheep fed diets containing silages of cactus pear combined with tropical forages. Forty sheep without defined breed, intact, with initial average weight of 22.65 ± 1.01 kg were distributed in a completely randomized design with 5 treatments and 8 replications. The experimental diets consisted of cactus pear silage (CPS), cactus pear + buffel grass silage (CPBS), cactus pear + gliricidia silage (CPGS), cactus pear + pornunça silage (CPPS), and corn silage (CS). CPGS provided higher water intake via food, total water intake, metabolic water, and excretion via feces and urine (P < 0.05). Animals that received diets containing CS showed higher water intake via drinking fountain, less efficient feeding and rumination of dry matter, less efficient rumination of neutral detergent fiber, grams of dry matter per cud, grams of neutral detergent fiber per cud, and the shortest average time spent in chewing per cud (P < 0.05). CPGS, CPPS, and CS provided longer times for rumination and numbers of cuds per day (P < 0.05). CPS showed animals spending more time in idleness, lower quantity of cuds per minute, higher concentration of crystals in urine, with a higher frequency of ammonia-magnesium phosphate and calcium oxalate. Silages based on cactus pear are an alternative to the supply of water via food for sheep in semi-arid.

Draft Genome Sequence of a New Fusarium Isolate Belonging to Fusarium tricinctum Species Complex Collected From Hazelnut in Central Italy.

In summer 2019, during a survey on the health status of a hazelnut orchard located in the Tuscia area (the province of Viterbo, Latium, Italy), nuts showing symptoms, such as brown-grayish spots at the bottom of the nuts progressing upward to the apex, and necrotic patches on the bracts and, sometimes, on the petioles, were found and collected for further studies. This syndrome is associated with the nut gray necrosis (NGN), whose main causal agent is Fusarium lateritium. Aiming to increase knowledge about this fungal pathogen, the whole-genome sequencing of a strain isolated from symptomatic hazelnut was performed using long Nanopore reads technology in combination with the higher precision of the Illumina reads, generating a high-quality genome assembly. The following phylogenetic and comparative genomics analysis suggested that this isolate is caused by the F. tricinctum species complex rather than F. lateritium one, as initially hypothesized. Thus, this study demonstrates that different Fusarium species can infect Corylus avellana producing the same symptomatology. In addition, it sheds light onto the genetic features of the pathogen in subject, clarifying facets about its biology, epidemiology, infection mechanisms, and host spectrum, with the future objective to develop specific and efficient control strategies.

A new inclusive MLVA assay to investigate genetic variability of Xylella fastidiosa with a specific focus on the Apulian outbreak in Italy.

The Olive Quick Decline Syndrome by Xylella fastidiosa subspecies pauca is among the most severe phytopathological emergencies nowadays. In few years, the outbreak devastated olive groves in Apulia (Italy), potentially endangering the entire Mediterranean basin. This research aimed to develop a multiple locus VNTR analysis assay, a molecular tool to differentiate between populations of the pathogen. It has already been successfully applied to different X. fastidiosa subspecies from various plant hosts. The previously published TR loci, together with a set of new design, have been tested in silico on the genome of the Apulian De Donno strain. The resulting selection of 37 TR loci was amplified on the genomic DNAs of the Apulian strains AND from representatives of X. fastidiosa subspecies, and directly on DNA extracted from infected plants. The assay clearly discerned among subspecies or even sequence types (ST), but also pointed out variants within the same ST so as to provide more detailed information on the dynamics and pathogen diffusion pathways. Its effective application even on total DNAs extracted from infected tissues of different host plants makes it particularly useful for large-scale screening of infection and for the strengthening of containment measures.

Black globe temperature from meteorological data and a bioclimatic analysis of the Brazilian Northeast for Saanen goats.

The black globe temperature (BGT) is not a common measurement for weather station networks, despite having great relevance to bioclimatic studies. The aim of this study was to propose equations for estimating the BGT, using meteorological data for different time scales and a bioclimatic evaluation of the Brazilian Northeast for breeding Saanen dairy goats. The data used in elaborating the equations were collected between 1 November 2014 and 31 October 2017. Data for BGT, incident global solar radiation (SR), air temperature (AT), relative humidity (RH) and wind speed were handled on a daytime, night-time, daily and monthly scale. One half of the database was used to adjust the equations and the other half in the evaluation. The bioclimatic diagnosis of the Brazilian Northeast was carried out based on mean monthly values of the black globe temperature and humidity index (BGHI) estimated for the four seasons of the year. For the daytime scale, an equation based on AT (BGT = 1.3897.AT-5.4421, r2 = 0.80) and a multiplicative model combining the effects of AT and SR (BGT = [1.3897.AT-5.4421] (0.0384.ln(SR)+0.7935], r2 = 0.91) were obtained. AT adjusted well for BGT on the night-time scale (BGT = 0.995.AT-0.6964, r2 = 0.99), daily scale (BGT = 1.1641.AT-1.5941, r2 = 0.97) and monthly scale (BGT = 1.1550.AT-1.3498, r2 = 0.98). The BGT can therefore be calculated from AT and/or SR for the daytime scale, and from AT only for the night-time, daily and monthly scales. In general, the west and centre-south of the state of Bahia offer the animals the most thermal comfort during each season of the year. In the state of Maranhão, heat stress occurs throughout the year, with the BGTI predominately in the range of 85-95. As such, strategies to combat heat stress should be encouraged to minimise the negative effects of climate on milk production in Saanen goats, and favour the milk-production chain in the northeast of Brazil.

Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies.

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Small RNA-Omics for Virome Reconstruction and Antiviral Defense Characterization in Mixed Infections of Cultivated Solanum Plants.

In plants, RNA silencing-based antiviral defense generates viral small RNAs (sRNAs) faithfully representing the viral genomes. We employed sRNA sequencing and bioinformatics (sRNA-omics) to characterize antiviral defense and to reconstruct the full genomic sequences and their variants in the evolving viral quasispecies in cultivated solanaceous plants carrying mixed infections. In naturally infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo-assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs, which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (genus Potexvirus) was tested for protection against strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically nontransmissible Southern tomato virus (genus Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA-generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes.

Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus.

The major threat for cassava cultivation on the Indian subcontinent is cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses which are bipartite begomoviruses with DNA A and DNA B components. Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) cause CMD in India. Two isolates of SLCMV infected the cassava cultivar Sengutchi in the fields near Malappuram and Thiruvananthapuram cities of Kerala State, India. The Malappuram isolate was persistent when maintained in the Madurai Kamaraj University (MKU, Madurai, Tamil Nadu, India) greenhouse, whereas the Thiruvananthapuram isolate did not persist. The recovered cassava plants with the non-persistent SLCMV, which were maintained vegetative in quarantine in the University of Basel (Basel, Switzerland) greenhouse, displayed re-emergence of CMD after a six-month period. Interestingly, these plants did not carry SLCMV but carried ICMV. It is interpreted that the field-collected, SLCMV-infected cassava plants were co-infected with low levels of ICMV. The loss of SLCMV in recovered cassava plants, under greenhouse conditions, then facilitated the re-emergence of ICMV. The partial dimer clones of the persistent and non-persistent isolates of SLCMV and the re-emerged isolate of ICMV were infective in Nicotiana benthamiana upon agroinoculation. Studies on pseudo-recombination between SLCMV and ICMV in N. benthamiana provided evidence for trans-replication of ICMV DNA B by SLCMV DNA A.