First report of three bunya-like viruses, apple luteovirus 1, and apple hammerhead viroid in apples from Hakkari, Türkiye.

Türkiye is a major apple fruit producer in the crossroads of Europe and the Middle East. Several reports have described the presence of multiple viruses affecting apple production in Türkiye, including apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV) (Kurçman 1977; Fidan 1994; Çaglayan et al. 2003). However, there are no reports of the presence of the recently discovered bunya-like viruses citrus concave gum-associated virus (CCGaV), and apple rubbery wood viruses 1 and 2 (ARWV1 and 2), as well as apple luteovirus 1 (ALV-1), and apple hammerhead viroid (AHVd) in Türkiye, all of which have been previously reported in other apple-producing countries (Wright et al. 2018; Liu et al. 2018; Zhang et al. 2014). Leaves from one Gala, two Granny Smith, and one Golden Delicious apple trees showing mild symptoms of curling, chlorosis, and yellowing were collected from four different orchards in the province of Hakkari, southeast Türkiye during June 2022 and sent to USDA APHIS Plant Germplasm Quarantine Program (under permit) for virus and viroid HTS-based diagnostics. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer's guidelines to prepare RNAseq libraries using the TruSeq Stranded Total RNA Library Plant Kit (Illumina, Inc) as described in Malapi-Wight et al. (2021). Libraries were sequenced on the NextSeq500 sequencer (PE 2x75), and approximately 45 million reads were obtained per each sample on average. Bioinformatic analysis was performed as described in Costa et al. (2022) using Phytopipe, where unclassified pathogen-derived reads were de novo assembled and contigs were compared to the NCBI viral nucleotide and protein databases by BlastN and BlastX respectively using a 10-4 e-value cutoff. Nearly complete genome contigs were obtained for ACLSV (OR640150) and ASPV (OR640151) in all four samples and for ASGV (OR640152) in 3 of the 4 samples. The average BlastN identity to sequences in GenBank was 92.3% for ACLSV, ranging from 89-94 %. BlastN identity for ASPV was 86%, ranging from 81-92 % while the ASGV average BlastN identity was 98.2%. Nearly complete genomes with average genome coverage of 92.4% and 95.6% for RNA1 and RNA2 of CCGaV (OR640153 and OR640154), were found in two of the four samples with BlastN identity of 94.7% and 94.8% to GenBank sequences. Additionally, nearly complete genome of the large (L), medium (M), and small (S) segments for ARWV1 were found in two samples with average genome coverage of 99.9%, 99.4%, and 100% respectively and BlastN identity of 98.8%, 95.2%, and 98.4% (OR640155, OR640156, OR640157). ARWV2 contigs were also found in 1 sample where M and S segments had a coverage of 99.8% and BlastN identity of 95.4% (OR640158 and OR640159). The nearly complete genome of ALV-1 was also found in two of four samples with genome coverage of 94.1% and an average BlastN identity of 93.4% (OR640160). AHVd was found in one of the Granny Smith trees with 19,260 mapped reads to the reference GenBank MH049335.1 and identity of 98.3% (OR640149). The HTS findings of CCGaV, ARWV1, ARWV2, and ALV-1, from Türkiye were later confirmed by Sanger sequencing using custom-designed primers targeting the coat protein, the RNA-dependent RNA polymerase, or ~390bp for the AHVd genome (Supplementary Table 1). To further learn about the incidence of these agents, we tested 12 other apple samples from six different neighboring orchards and found them at 18.8% rate for CCGaV, 12.5% for both ARWV1 and ARWV2, 25% for ALV-1, and 37.5% for AHVd respectively. To our knowledge, this is the first report of the apple viruses CCGaV, ARWV1, ARWV2, and ALV-1, and the AHVd viroid in Türkiye. Further studies of the impact of these agents on orchard's health are necessary, including their prevalence in high apple production regions of Türkiye.

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

Different loci control resistance to different isolates of the same race of Colletotrichum lindemuthianum in common bean.

KEY MESSAGE: Linkage and genome-wide association analyses using high-throughput SNP genotyping revealed different loci controlling resistance to different isolates of race 65 of Colletotrichum lindemuthianum in common bean. Development of varieties with durable resistance to anthracnose is a major challenge in common bean breeding programs because of the extensive virulence diversity of Colletotrichum lindemuthianum fungus. We used linkage and genome-wide association analyses to tap the genomic regions associated with resistance to different isolates of race 65. Linkage mapping was done using an F2 population derived from the cross between the Mesoamerican common beans BRS Estilo x Ouro Vermelho, inoculated with two different isolates of race 65. Association genetics relied on a diversity common bean panel containing 189 common bean accessions inoculated with five different isolates of race 65 as an attempt to validate the linkage analysis findings and, eventually, identify other genomic regions associated with resistance to race 65. The F2 population and diversity panel were genotyped with the BARCBean6K_3 Illumina BeadChip containing 5398 SNP markers. Both linkage and genome-wide association analyses identified different loci controlling resistance to different isolates of race 65 on linkage group Pv04. Genome-wide association analysis also detected loci on Pv05, Pv10 and Pv11 associated with resistance to race 65. These findings indicate that resistance to race 65 can be overcome by the virulence diversity among different isolates of the same race and could lead to the loss of resistance after cultivar release. We identified 25 resistant common bean cultivars to all five isolates of race 65 in the diversity panel. The accessions should be useful to develop cultivars combining different resistance genes that favor durable resistance to anthracnose in common bean.

Are duplicated genes responsible for anthracnose resistance in common bean?

The race 65 of Colletotrichum lindemuthianum, etiologic agent of anthracnose in common bean, is distributed worldwide, having great importance in breeding programs for anthracnose resistance. Several resistance alleles have been identified promoting resistance to this race. However, the variability that has been detected within race has made it difficult to obtain cultivars with durable resistance, because cultivars may have different reactions to each strain of race 65. Thus, this work aimed at studying the resistance inheritance of common bean lines to different strains of C. lindemuthianum, race 65. We used six C. lindemuthianum strains previously characterized as belonging to the race 65 through the international set of differential cultivars of anthracnose and nine commercial cultivars, adapted to the Brazilian growing conditions and with potential ability to discriminate the variability within this race. To obtain information on the resistance inheritance related to nine commercial cultivars to six strains of race 65, these cultivars were crossed two by two in all possible combinations, resulting in 36 hybrids. Segregation in the F2 generations revealed that the resistance to each strain is conditioned by two independent genes with the same function, suggesting that they are duplicated genes, where the dominant allele promotes resistance. These results indicate that the specificity between host resistance genes and pathogen avirulence genes is not limited to races, it also occurs within strains of the same race. Further research may be carried out in order to establish if the alleles identified in these cultivars are different from those described in the literature.

Growth parameters impairment in patients with food allergies.

Background and Aims. Food allergy (FA) is a common disease that is rapidly increasing in prevalence for reasons that remain unknown. Objective. The aim of this study was to analyze the clinical characteristics and anthropometric data of patients with food allergies followed in a tertiary centre of allergy and immunology. Methods. A retrospective study was performed that assessed the data records of patients with food allergy diagnosis, covering a period from February 2009 to February 2012. Results. 354 patients were evaluated in the period; 228 (69.1%) patients had a confirmed FA diagnosis. The z-scores for weight-for-age, height-for-age, and body mass indices-for-age showed lower significant values in the FA group compared with the non-FA group by Mann-Whitney test, with significance values of P = 0.0005, P = 0.0030, and P = 0.0066, respectively. There were no statistical differences in sex, gestational age, birth type, breastfeeding period, and age of introduction of complementary formulas based on cow milk protein between groups. Conclusion. FA patients had a lower growth rate in comparison with patients without FA. The early recognition of food allergies with the establishment of protein-implicated diet exclusion, in association with an adequate nutrient replenishment, is important to reduce the nutritional impact of food allergies.