Symptomatic Samples Research Articles

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy.

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy Wassim Habib1, Mariangela Carlucci2, Roberto Fasano3, Franco Nigro1,2 † 1 Centre of Research, Experimentation and Training in Agriculture (CRSFA) - Basile Caramia, via Cisternino 281, Locorotondo, 70010 Bari, Italy 2 Department of Soil, Plant and Food Sciences, University of Bari - Aldo Moro, via Amendola 165A, 70126 Bari, Italy 3 Bioricerche, Phytopathological Lab, via Pio XI 30, Bellizzi, 84092 Salerno, Italy † Corresponding author: F. Nigro. Email: franco.nigro@uniba.it Keywords: Phoma-like, Multi-locus gene sequencing, Brassica oleracea L. var. botrytis, Campania. In September 2022, a severe foliar disease was observed on 2-3-week-old 'Corsaro' and 'Parthenon' seedlings of cauliflower (Brassica oleracea L. var. botrytis) grown under greenhouses in a nursery in Caserta province, Campania (Italy), with an incidence of 90% on about 150,000 plants. Affected plants showed numerous small, irregular, and depressed lesions bordered by a chlorotic halo on leaves that become necrotic, and later drop out giving a shot-hole effect (Fig. 1). Isolations were performed on leaf portions from 30 symptomatic samples which were surface-sterilised by dipping in 70 % ethanol for 30s, then in a sodium hypochlorite solution (1%) for 30s, and rinsed two times in distilled sterilised water. Tissue fragments were left to dry on sterile filter paper and then seeded on potato dextrose agar (PDA) amended with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich S.R.L, Steinheim, Germany). After 6 to 8 days of incubation at 24 ± 1°C, colonies revealed a consistent growth of a Phoma-like fungus resembling Stagonosporopsis genus. On PDA, one-week-old colonies were dark olivaceous green to brown with white regular margin (Fig. 2B). Pycnidia (32-160 µm × 30-95 µm) were subglobose, and conidia (2.7-4.8 µm × 1-2.7 µm) ellipsoidal, with rounded ends, aseptate, with two polar guttules (Fig. 2C). All isolates had similar morphological features, therefore further analyses were performed on one representative strain (CRSFA.753.22). The multilocus phylogenetic approach using Maximum likelihood method and Tamura-Nei model on rDNA-ITS, TUB and RPB2 sequences (accession numbers: OQ318550, OQ326503, OQ326504) including reference strains of Stagonosporopsis (Dong et al. 2021) demonstrated that CRSFA.753.22 clustered with Stagonosporopsis pogostemonis Luo, Huang & Manawas holotype (ZHKUCC 21-0001) with 91% bootstrap support. ITS and RPB2 sequences had 100% BLAST match to ZHKUCC 21-0001, whereas TUB sequence had 99.69%. Pathogenicity tests were performed on 20 healthy 'Corsaro' plantlets. Leaves were surface sterilized with 70% alcohol and wounded with a sterile tip of a needle. A suspension of conidia and mycelial fragments, obtained from a 10-days-old colony of the strain CRSFA.753.22, was sprayed on the leaf surfaces at a distance of 20 cm. Five controls plants were inoculated with sterile distilled water. Plants were then covered with plastic bags and kept on the shelf in a growth chamber at 25° C, with a 12-h photoperiod. The first symptoms developed on leaves five days post-inoculation as irregular small brown spots, that were gradually expanded. Leaf tissues showed chlorosis which evolved into necrosis (Fig. 2A). Stagonosporopsis pogostemonis was consistently re-isolated from all diseased leaves, but not from control plants, thus fulfilling Koch's postulates. In Italy, Stagonosporopsis species have been reported as severe plant pathogens (Garibaldi et al. 2022; Guarnaccia et al. 2022), and this is the first report of the species S. pogostemonis in the country. References Dong, Z.Y., et al. 2021. Pathogens 10:1093. doi: 10.3390/pathogens10091093 Garibaldi, A., et al. 2022. Plant Pathol. J. 104:1157. doi: 10.1007/s42161-022-01138-7 Guarnaccia, V., et al. 2022. Plant Pathol. J. 104:1491. doi: 10.1007/s42161-022-01197-w.

Assessment of genetic diversity of latent bacteria in coconut leaves associated with lethal yellowing disease symptoms in Kenya

AbstractCoconut (Cocos nucifera) is an economically important palm tree with diverse applications. However, limited scientific research exists on coconut diseases in the Kenyan coastal region. This cross‐sectional study aimed to investigate the genetic diversity of latent bacteria associated with yellowing symptoms in coconut plants along the Kenyan coast. Sixty‐two samples with symptoms were collected and their bacterial diversity assessed using culture‐independent methods. DNA was extracted from controls and symptomatic samples using the cetyl trimethyl ammonium bromide (CTAB) method. Next‐generation sequencing with Illumina MiSeq was used to profile the bacterial communities using amplicons of 16S ribosomal RNA sequences (V4 region). Sequence data were analysed using the Quantitative Insights Into Microbial Ecology 2 (QIIME 2) pipeline. A total of 113,330 reads were obtained, which clustered into 285 Operational Taxonomic Units. Bacterial diversity was highest in Kilifi, followed by Kwale and Lamu, while control samples exhibited low bacterial diversity. Actinobacteria was the predominant phylum across all counties, and Streptomyces was the most abundant genus. Kilifi and Kwale counties were more affected than Lamu. This is a pioneer study that provides insight into the bacterial diversity associated with yellowing disease symptoms in coconut plants in Kenya and will help with future elucidation of the agents causing or exacerbating coconut disease symptoms.

First Report of Mixed Infection of Jasmine Mosaic-Associated Virus (JMaV) and Jasmine Virus H (JaVH) in Jasmine Species in Florida, United States

Over the past 24 years, virus-like symptoms have been observed on Jasminum spp. in Florida. In September 2018, symptomatic Angelwing jasmine leaves from a hedge in Alachua, Florida, were tested for jasmine mosaic-associated virus (JMaV) and jasmine virus H (JaVH). Identification of expected size amplicons was confirmed by sequencing (accession numbers MN245037, MT559755). In December 2018, symptomatic samples from two Gold Coast jasmine plants from St. Lucie County, Florida, were similarly tested. Only JaVH tested positive (accession number MN442626). In July 2019, Angelwing jasmine hedge in Orange County, Florida, exhibiting similar symptoms tested positive only for JaVH (GenBank accession number MN442627). In April 2023, samples from a symptomatic Gold Coast jasmine hedge in Indian River County, Florida, also tested positive only for JaVH. In May 2023, a symptomatic Angelwing jasmine sample from Miami-Dade County tested positive for both JMaV and JaVH. This is the first report of JMaV and JaVH in single or mixed infections in jasmine in Florida, adding to the known virus species infecting jasmine in the state. Because jasmine is propagated vegetatively, it is likely that dissemination of cuttings made from infected plants is responsible for widespread distribution of these viruses.

The first survey using high-throughput sequencing of cereal and barley yellow dwarf viruses in Irish spring and winter barley crops

Yellow dwarf viruses (YDVs) are the most economically important plant viruses impacting cereal production worldwide and include viruses from the genus Luteovirus (e.g., barely yellow dwarf virus (BYDV)-PAV, BYDV-PAS, BYDV-MAV, BYDV-kerII, BYDV-kerIII) and Polerovirus (e.g., cereal yellow dwarf virus (CYDV)-RPV, CYDV-RPS). Until now, much of our knowledge on YDVs infecting Irish barley crops (Hordeum vulgare L.) has come from serological assays; however, due to cross-reactivity it can be difficult to discriminate between viruses of different species. In this study, we have carried out a high-throughput sequencing survey of symptomatic crops, positive with serological assays, to identify YDVs infecting Irish spring and winter barley crops and establish reference genomes to support further development of molecular surveillance tools. In total, RNA was extracted from 45 symptomatic crop samples that were collected across Ireland over 2 yr and sequenced following rRNA depletion. Three samples of barley plants from BYDV-infected aphid colonies were also included. BYDV-MAV was identified in all field samples sequenced. This confirms previous evidence based on serological assays that BYDV-MAV is the dominant YDV in Irish barley crops. We have also identified BYDV-PAS in 29% of symptomatic field samples, the first report of this species in Ireland. In addition, BYDV-PAV was also found, and crop samples with mixed infections were common; although in mixed infections the greatest proportion of YDV reads originated from BYDV-MAV. Finally, CYDV-RPS, the more severe variant of CYDV-RPV belonging to the genus Polerovirus, was identified in a single sample. The complete genomes, assembled from this first sequence-based survey, will enable the development of molecular surveillance tools with greater virus specificity, to further support the Irish aphid and YDV monitoring network.

Identification of Gonatophragmium mori Causing Mulberry Zonate Leaf Spot Disease and Characterization of Their Biological Enemies in Guangxi, China.

Mulberry zonate leaf spot disease (MZLSD) is an important fungal disease of mulberry trees, which seriously affects the productivity and quality of mulberry leaves. MZLSD has been widely reported in sericultural production areas in Guangxi, China, in recent years. In this study, the causal agent of MZLSD was isolated from symptomatic samples and identified as Gonatophragmium mori (Acrospermaceae) based on morphological characterization and molecular analyses using nucleotide sequences of the internal transcribed spacer (ITS) and large subunit ribosomal DNA (LSU rDNA). Pathogenicity tests confirmed that G. mori is the pathogen responsible for MZLSD. Furthermore, we isolated antagonistic endophytic bacteria (AEB) from healthy mulberry leaves. Plate confrontation experiments showed that the lipopeptide crude extracts (LPCE) of three endophytic bacteria can inhibit the growth of G. mori, and the diameter of the antibacterial circle reaches more than 60 mm when their concentration of LPCE is 200 mg/ml. Light microscopy and scanning electron microscopy revealed that LPCE caused drastic changes in mycelial morphology. Fluorescence microscopy and transmission electron microscopy showed that the LPCE-induced apoptosis-like cell death in G. mori hyphae. Finally, based on morphological and molecular features, we identified the three isolates as Bacillus subtilis DS07, B. subtilis DS32, and B. velezensis Q6, respectively. To our knowledge, this is the first time to identify G. mori by combining characterization and molecular analyses, and we provide timely information about the use of biocontrol agents for suppression of G. mori.

First Detection of Cucurbit Yellow Stunting Disorder Virus in Iraq

During the growing season of 2022, courgette plants displaying foliar yellowing that included leaf mottle and interveinal chlorosis along with green veins were observed in numerous courgette fields of Al-Yusufiyah, Baghdad Province, Iraq. Bemisia tabaci was abundant with the foliar yellowing plants in all fields investigated. Fifty symptomatic leaf samples showing typical foliar yellowing were collected randomly from several courgette fields. Three representative samples were sent to Macrogen (Seoul, South Korea) for high-throughput RNA sequencing. The BLASTn analyses of the assembled genome of cucurbit yellow stunting disorder virus (CYSDV) isolate Iraq-1 segment RNA1 and RNA2 demonstrated pairwise nucleotide identity between 99.23 and 99.84% for segment RNA1 and between 97.58 and 97.92% for segment RNA2 with several corresponding global isolates of CYSDV. These two segments of the CYSDV isolate Iraq-1 were deposited in GenBank under accession numbers OQ685958.1 and OQ685959.1, respectively. Additionally, a phylogenetic analysis indicated a relationship between CYSDV isolate Iraq-1 and numerous isolates of CYSDV reported from various origins, mainly from the United States (MW147553.1 and EF547827.1) and Spain (AJ537493.1 and NC_004809) for the segment RNA1. However, the segment RNA2 of CYSDV isolate Iraq-1 was close to those from Spain (NC_004810.1, AY242078.1, and AJ439690.2), Greece (OL584360.1), and the United States (FJ492808.1). Although CYSDV was previously reported in Europe, such as in Spain and France; in Asia in Iran and Jordan; and in North America in the United States and Mexico, it has not been reported in Iraq. Thus, this is the first report of CYSDV in Iraq.

Detection and molecular identification of Entamoebaspecies in faecal samples from Duhok province, Kurdistan Region, Iraq.

The study involved the estimation of the prevalence of Entamoeba spp. using microscopy and molecular techniques among symptomatic outpatients during April 2021 to March, 2022. Stool samples were collected from 2592 outpatients with amoebiasis symptoms of both sexes and different ages (≤ l to 60). Also, 107 stool samples were taken randomly from asymptomatic individuals and examined microscopically to detect infection with Entamoeba spp. the positive specimens were used for molecular analysis with positive symptomatic samples targeting the 18S rRNA gene by nested PCR. Microscopically 21.68% (562/2592) were positive, for Entamoeba spp. Males showed highest infection rate than females (67.43% vs 32.56%). Ages from 1-10 years showed the highest rate (54.09%), and urban inhabitant had somewhat a higher rate than rural one (58.54% vs 41.45%) which was statistically non-significant(P>0.05). Among asymptomatic individuals, 57% (61/107) were positive for Entamoeba spp. Nested PCR analysis yielded 73% positive samples for Entamoeba spp. with a fragment size of 897 bp. Three fragment sizes were produced, for E. histolytica, E. dispar and E. moshkovskii which were 439, 174 and 553 bps, respectively. Single infection occurred with, E. histolytica in 46%, of symptomatic and 6% of asymptomatic cases, E. dispar in 38% of asymptomatic and 10% of symptomatic cases, E. moshkovskii, reported at very low rate among both groups.

Pathogenicity of Botryosphaeriaceae and Phytophthora species associated with Paulownia dieback, canker and root rot in Italy

In recent years, an unusual decline and mortality has been observed in Paulownia plantations throughout the Marche region (Central Italy). Given the economic importance of this emerging forest crop, a study was conducted to determine which pathogens are directly involved in this syndrome. Field surveys performed in two plantations revealed the widespread occurrence of severe disease symptoms such as leaf chlorosis, crown thinning, shoot and branch dieback, sunken cankers, epicormic shoots and root rot. Disease incidence was also assessed by aerial remote sensing (RS) technologies using drones. Symptomatic samples collected from both stem and root tissues yielded fungal and fungal-like colonies representing two distinct families: Botryosphaeriaceae and Peronosporaceae. Morphological and DNA sequence data revealed five distinct species, identified as Macrophomina phaseolina and Botryosphaeria dothidea (Botryosphaeriaceae), Phytophthora pseudocryptogea, P. citrophthora and P. erythroseptica (Peronosporaceae). Given that all species are reported here for the first time on Paulownia, Koch’s postulates were satisfied inoculating the three Phytophthora species and two Botryosphaeriaceae at the collar of the stem of potted 1-year-old rooted cuttings in June 2023. Thirty days after inoculation, all plants showed the same symptoms as those observed in the field.

First report of a 'Candidatus Phytoplasma australasiaticum'-related phytoplasma strain associated with shoot proliferation disease of variegated croton in Taiwan.

Codiaeum variegatum (family Euphorbiaceae) is a leaf ornamental commonly known as variegated croton, which is often found in gardens or grown as indoor plants. In December of 2022, two cutting-propagated variegated croton plants exhibiting abnormal shoot proliferation and little leaf symptoms (Fig. S1) were found in a nursery owned by a private breeder in Wanluan Township, Pingtung County, Taiwan. The plants were propagated from a single stock plant, which died during transplanting from a commercial nursery in Changzhi Township, Pingtung County. To determine the potential cause of such symptoms, leaf tissues were collected from the center of the two symptomatic plants. Their DNA were extracted with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, NJ) and used for further testing. As controls, two symptomless stock plants were collected from the commercial nursery in Changzhi Township and used to produce cutting-propagated plants; leaf DNA was extracted from each plant as described above. The DNA samples were subjected to PCR testing using the phytoplasma-specific primer pair P1/P7 (Schneider et al. 1995), and only DNA from the symptomatic plants produced the expected 1.8-kb amplicon. The two phytoplasma isolates detected in the plants were designated as CvaA and CvaB. After sequencing and analyzing the data using the iPhyClassifier program (Zhao et al. 2009), both CvaA and CvaB were classified to subgroup 16SrII-A (GenBank accession no. L33765) with a similarity coefficient of 1.0. The near-full-length 16S rDNA fragments of the detected isolates (GenBank accession no. OR794242) were also identical (1,463/1,463 bp) to that of NCHU2014 (GenBank accession no. CP040925, bp 537768-539230), a reference 'Ca. Phytoplasma australasiaticum'-related strain (16SrII-A) found in Taiwan (Chang et al. 2015; Rodrigues Jardim et al. 2023). To validate the results, the DNA samples were also tested with 16SrII group-specific semi-nested PCR targeting the elongation factor Tu gene. The outer and inner primer pairs used were TUF-II-F1/TUF-II-R1 and TUF-II-F2/TUF-II-R1, respectively (Al-Subhi et al. 2017). An expected amplicon was detected in the symptomatic samples but not in the symptomless counterparts. The amplified fragments' sequences (GenBank accession no. OR634931) were identical to that of the elongation factor Tu gene of the 'Ca. Phytoplasma australasiaticum'-related strain NCHU2014 (989/989 bp; GenBank accession no. CP040925, bp 139344-140332). The protein translocase gene secY of the detected phytoplasma was also amplified and sequenced using semi-nested primers SecYF1(II), SecYF2(II) and SecYR1(II) (Lee et al. 2010). Again, the sequences of the detected isolates (GenBank accession no. OR862773) were identical to that of NCHU2014 (1,263/1,263; GenBank accession no. CP040925, bp 192846-194108). The quality of the DNA samples was confirmed by PCR targeting the plant host's 28S rDNA using primer pair 28KJ/28C (Cullings 1992) and all symptomatic and symptomless samples produced the target amplicon (0.7 kb). 16SrII phytoplasmas have been detected in different host plants in Taiwan (Chang et al. 2015). To our knowledge, this is the first record of this group of pathogens infecting variegated croton in Taiwan. Branch-inducing phytoplasma has been used to improve the ornamental values of poinsettias, another Euphorbiaceae species (Lee et al. 1997). Further testing is needed to determine whether the phytoplasma detected in this work could be used for similar purposes.

Distribution of Papaya Ring Spot Virus Infecting Papaya in Kerala, India

In the midst of the rising interest of papaya cultivation in Kerala, it is inevitable to study the current status of the Papaya ring spot disease in the state. Knowing the severity and distribution of PRSV isolates, causing ring spot disease, helps in the better formulation of effective management strategies against it. Purposive sampling surveys carried out in 2021-22 across five agro ecological units (AEUs) of Kerala, recorded papaya ring spot disease incidence (PRSD) ranging from 50 to 100 per cent in the cultivated areas with vulnerability index of 33.54 to 98.22. Highest disease incidence was recorded from AEU 8- Southern Laterites: Thiruvananthapuram (100.0%) and highest vulnerability index was recorded from Kalliyoor (98.22). Twenty symptomatic samples tested positive for PRSV in double antibody sandwich- ELISA (DAS-ELISA) using PRSV polyclonal antiserum. RT-PCR using coat protein gene specific primers RKJ 52 and 3 yielded amplicons of size approximately ~890bp in all the samples. Virulence of the Kerala isolates was evaluated based on the symptom expression, disease incidence and vulnerability index, upon mechanical transmission of PRSV on two months old papaya plants (variety- Red Lady). PRSV isolate from Kalliyoor (TVM1) inoculated on papaya plants expressed chlorosis, mottling, malformation of leaves and stunting with 96.80 vulnerability index. Maximum vulnerability index and severe symptoms including stunting in the inoculated plants were observed in isolates from southern and central Kerala, which include Thiruvananthapuram, Alappuzha and Thrissur had the most virulent PRSV isolates compared to north Kerala.

Revealing microbial patterns in the rhizosphere of pecan trees asymptomatic and symptomatic for Texas root rot using a high-throughput sequencing approach

The rhizosphere harbors diverse microbial communities that play crucial roles in nutrient cycling and plant health. Understanding the composition and dynamics of these communities is essential for optimizing agricultural productivity and disease management. In this study, bacterial and fungal communities in the rhizosphere of pecan trees (Carya illinoinensis) asymptomatic and symptomatic for Texas root rot were characterized using high-throughput sequencing of the 16S rRNA gene and ITS region. The results indicated that Proteobacteria and Actinobacteria were the predominant bacterial phyla in both conditions. The predominant fungal phyla comprised Basidiomycota and Ascomycota in symptomatic conditions and Ascomycota and Mortierellomycota in asymptomatic ones. At the genus level, Streptomyces and Pseudomonas were the most abundant bacterial genera; among fungi, Scleroderma and Psathyrella were predominant in symptomatic samples and Mortierella in asymptomatic samples. α-diversity analysis showed significant differences in the fungal community. Specific bacterial and fungal biomarkers for asymptomatic and symptomatic conditions were identified, highlighting their importance in the pecan tree rhizosphere. A total of six bacterial and seven fungal taxa were detected in symptomatic samples, while four bacterial and 16 fungal taxa were detected in asymptomatic ones. These included genera known for their roles in soil health, such as Pseudomonas, Kaistobacter, Streptomyces, Psathyrella, Scleroderma, and Mortierella. Co-occurrence network analysis revealed higher complexity and interaction levels among microbial communities in asymptomatic samples, suggesting a healthier network supporting microbial communities. This study provides comprehensive and valuable insights into microbiome dynamics and microbial resources for future studies aimed at improving agricultural production through the study of rhizosphere microorganisms.

Sensitivity and Specificity of the Boston Criteria Version 2.0 for the Diagnosis of Cerebral Amyloid Angiopathy in a Community-Based Sample.

The Boston criteria are a set of clinical and neuroimaging features that enable accurate diagnosis of cerebral amyloid angiopathy (CAA) without invasive methods such as brain biopsies or autopsy. The last updates to the Boston criteria, named version 2.0, were recently released and incorporated new nonhemorrhagic MRI features. These criteria have been validated in symptomatic samples, with improved diagnostic yield. We set out to investigate the accuracy of the Boston criteria v2.0 for the diagnosis of CAA in a community-based sample. Participants were recruited from longitudinal clinical-pathologic studies of aging conducted at the Rush Alzheimer's Disease Center in Chicago: the Religious Orders Study and the Rush Memory and Aging Project. Deceased participants with in vivo 3T MRI and detailed pathologic data available were included in the analysis. We compared the diagnostic yield of the current and earlier versions of the Boston criteria in our sample. Among those classified as probable CAA according to the Boston criteria v2.0, we investigated the ability of each neuroimaging marker to distinguish between false-positive and true-positive cases. In total, 134 individuals were included in the study (mean age = 82.4 ± 6.0 years; 69.4% F), and 49 of them were considered pathology-proven definite cases with CAA (mean age = 82.9 ± 6.0 years; 63.3% F). The Boston criteria versions 1.0 and 1.5 yielded similar sensitivity (26.5%, both), specificity (90.6% and 89.4%, respectively), and predictive values (negative: 68.1% and 67.9%; positive: 61.9% and 59.1%, respectively). The recently released Boston criteria v2.0 offered higher sensitivity (38.8%) and slightly lower specificity (83.5%). Among those classified as probable CAA (v2.0), pathology-proven true-positive cases had higher numbers of strictly cortical lobar microbleeds compared with false-positive cases (p = 0.004). Similar to findings from symptomatic samples, the inclusion of nonhemorrhagic neuroimaging markers in the updated Boston criteria offered a 12.3% gain in sensitivity among community-dwelling individuals, at the expense of a 5.9% drop in specificity. In cases with probable CAA, the cortical location of microbleeds may represent a promising distinguishing feature between true-positive and false-positive cases. Despite its improved performance, the diagnostic sensitivity of the updated criteria in a community-based sample remains limited. This study provides Class II evidence that the Boston criteria v2.0 accurately distinguishes people with CAA from those without CAA.

Eucalyptus leaf spot disease caused by Coniella eucalyptorum in Sri Lanka

Coniella has a wide distribution as a plant pathogen in Eucalyptus species. Here we report a leaf spot disease caused by Coniella eucalyptorum on Eucalyptus camaldulensis in Sri Lanka. The pathogen was isolated from symptomatic leaf samples of E. camaldulensis plants from Polonnaruwa District in Sri Lanka. The isolated fungus was identified as C. eucalyptorum based on morphological characteristics and multi-gene phylogenetic analysis of combined sequences of the internal transcribed spacer region (ITS), the partial translation elongation factor 1-α gene (tef1-α), and large subunit of nuclear ribosomal RNA (LSU). The pathogenicity test was performed to confirm its pathogenicity to the host, which produced symptoms similar to those observed in the field. This is the first report of C. eucalyptorum associated with Eucalyptus plantations in Sri Lanka. This finding is important to expand the knowledge of plant pathogen diversity in Eucalyptus species and other economically important crops in Sri Lanka.

Survey of Soilborne Pathogens Infecting Strawberry in Santa Maria, California

ABSTRACTTo determine the prevalence of the four major soilborne pathogens of strawberry in the Santa Maria growing district in California, symptomatic plant samples (n = 100) were collected from 68 fields between May and September 2022. Samples comprised 28.3% of 2021 fall-planted strawberry fields and 14.0% of 2022 summer-planted strawberry fields in the district. Recombinase polymerase amplification was used to detect Macrophomina phaseolina, Fusarium oxysporum f. sp. fragariae, Verticillium dahliae, and Phytophthora spp. in plant crown material. Macrophomina phaseolina was most prevalent, detected in 52 samples, followed by Verticillium dahliae in 17 samples, F. oxysporum f. sp. fragariae in 16 samples, and Phytophthora spp. in 14 samples. Macrophomina phaseolina was more likely in eastern field locations (P = .0159) and in instances where strawberries were planted consecutively (P = .0100). Disease incidence was more likely to be higher in samples from organic fields than conventional fields (P = .0304) and in samples from flat-fumigated fields than fields fumigated in-line (P = .0124).

Molecular Identification and Fungal Diversity Associated with Diseases in Hass Avocado Fruit Grown in Cauca, Colombia.

Hass avocado fruit diseases are one of the main marketing constraints in Colombia. To identify and reveal the diversity of fungi associated with diseases in fruits and peduncles, symptomatic samples were collected from 67 farms in the 9 main Hass avocado-producing municipalities of the department of Cauca located in southwestern Colombia. A total of 237 monosporic isolates were obtained that were subjected to DNA extraction, amplification of the ITS region, sequencing and functional diversity analysis based on Hill numbers by municipality and altitude. The results indicated that the genera Pseudocercospora, Diaporthe, Colletotrichum, Neofusiococcum, Lasiodiplodia and Pestatoliopsis were associated with fruit diseases. The genus with the highest number of effective species was Colletotrichum. The ITS region revealed 100% identity of the species Pseudocercospora norchiensis, which was the first report of this pathogen in the crop. There was a greater richness and diversity of associated species in the three municipalities, revealing that the higher the altitude was, the lower the richness and diversity of fungi associated with fruit diseases. These results will provide a better understanding of the identification and diversity of pathogenic microorganisms present in avocado production systems in this region of Colombia.

Identification, Characterization, and Pathogenicity of Fungi Associated with Strawberry Fruit Rot in Shandong Province, China.

China is the largest strawberry producer and exporter worldwide and has been constantly challenged by fruit rot diseases in recent years. Symptoms of various diseases on strawberry fruits were observed in Huangqiyuan Base, an important strawberry-producing region in Shandong Province, and symptomatic samples were collected from January to April 2021 for follow-up studies. In the present study, 137 isolates were obtained and classified into nine species based on morphological characteristics and multilocus phylogenetic analysis (ITS, GAPDH, HIS3, RPB2, EF-1α, HSP60, G3PDH, and/or TUB2), namely, Botrytis cinerea, B. fabiopsis, Alternaria alternata, A. tenuissima, Fusarium proliferatum, F. graminearum, F. ipomoeae, F. incarnatum, and Colletotrichum siamense. Pathogenicity results suggested that all nine pathogenic species could induce fruits to exhibit symptoms similar to those naturally infected in fields. The symptoms around the inoculation points varied, including dense white mycelia caused by Botrytis spp., fading and depression caused by Fusarium spp., black-brown rot caused by Alternaria spp., and shrinkage and dehydration caused by Colletotrichum spp. Overall, B. cinerea was the dominant pathogen, accounting for 61.3% of the total isolates, and showed significantly higher virulence against strawberry fruits than other species. In addition, this is the first report to identify B. fabiopsis, A. alternata, A. tenuissima, F. proliferatum, F. graminearum, F. ipomoeae, and F. incarnatum as causal agents of strawberry fruit rot in Shandong Province, China.

Near infrared spectroscopy as a novel non-invasive tool for the detection of lactococcosis in rainbow trout

Lactococcosis, mainly sustained by the warm-water bacterium Lactococcus garvieae, is a disease associated with evident mortalities and economic lossed. Main disease signs are commonly bilateral exophthalmia, hyperpigmentation in the body, haemorrhages around the eye area, opercula, and mouth region, swollen abdomen, and anal prolapse. An effective monitoring system of fish diseases relies on rapid techniques for the timely management of infection outbreaks. We evaluated the potential of the SCiO sensor, a portable near infrared spectroscope, to assess in field the health status of rainbow trout. The study sample was composed of 20 symptomatic and 20 asymptomatic rainbow trout obtained from an aquaculture farm and submitted to molecular detection of L. garvieae and SCiO examination. In order to detect presence or absence of septicaemia condition, blood smears were prepared from peripheral blood. Part of the symptomatic individuals (9/20) showed septicaemia, whereas no bacteria were found in the asymptomatic individuals. PCR assay of bacterial DNA was positive in spleen and kidney of 19/20 symptomatic and in 1/20 asymptomatic individuals. Bacteriological analysis revealed L. garvieae in 18/20 symptomatic samples and in 2/20 asymptomatic samples. The SCiO sensor was found to be able to discriminate between healthy and sick fish [sensibility 0.95 (0.75–1.00 I.C. 95%), specificity 0.80 (0.56–0.94 I.C. 95%)]. By virtue of rapid acquisition, low cost and use on site, the SCiO was considered as a useful diagnostic tool for monitoring L. garvieae infection in rainbow trout.

Differential occurrence and strain composition of potato viruses between two major seed potato production zones in China suggest the need for different management strategies for the zones

Viral disease is one of the major reasons leading to yield and quality reduction of potato production. To gain better knowledge about seed potato quality in China, the occurrences of six common viruses, including potato leafroll virus (PLRV) and potato viruses A (PVA), M (PVM), S (PVS), X (PVX) and Y (PVY), were investigated in two of China’s four potato production zones (specifically, Zone I and Zone IV) from 2013 to 2016. A total of 1231 random tuber samples (Zone I, n = 571; Zone IV, n = 660) and 532 symptomatic leaf samples (Zone I, n = 375; Zone IV, n = 157) were collected in seed potato and commercial potato fields and tested for the viruses by reverse transcription (RT)-PCR (tuber samples) and/or enzyme-linked immunosorbent assay (ELISA; leaf samples). The results showed that virus-free tubers accounted for 79.7% and 52.0% in seed tuber samples from zones I and IV, respectively. All of the six viruses analysed were present in both zones I and IV, but occurred at different frequencies, with PVY and PVS being the predominant viruses in zones I and IV, respectively. Moreover, 44.0% and 39.8% of the virus-positive samples in zones I and IV, respectively, were found to be infected with more than one virus. Strain typing of PVS and PVY demonstrated the existence of similar strains of PVY (i.e. PVYO, PVYNTNb, PVYN:O, PVYN- Wi, PVY – (SYR-I and -II) and of PVS (i.e. PVSO and PVSA) in both zones. For PVY, recombinant strains were predominant, with SYR-II (27.7%) being the most abundant strain in Zone I and SYR-I (53.3%) being the predominant strain in Zone IV. PVSO was the predominant strain in Zone I whereas PVSA was the dominant strain in Zone IV. This study revealed not only the high incidence of viruses in both seed and commercial potatoes in China, but also the differential occurrence and strain composition of the viruses between two major production zones in China for the first time. In addition, the study suggests the need for different management strategies for different production zones to enhance the quality of seed potatoes in China.

Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive (Olea europaea) Twigs.

Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as Cladosporium, Didymosphaeria, Paraconiothyrium, Penicillium, Phoma, Verticillium, and others.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 μm x 3.77-6.84 μm (average 22.90 μm x 5.20 μm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.