First Report of Phytophthora parsiana Causing Crown and Root Rot on Guayule in the United States

Guayule (Parthenium argentatum A. Gray) is a perennial shrub plant (approximately 50 cm in height) cultivated in the southwestern United States. It produces natural low-allergenic latex, resins and high-energy biofuel feedstock. During August 2021, a crown and root rot disease was observed on 2-year-old plants of direct-seeded guayule cultivar ‘Az 2’ in research plots located in Pinal county, Arizona, where a record 36 cm of rainfall fell during monsoon season. Symptoms included yellowing of leaves, wilting, and plant death. Average disease incidence was 16%. Isolation from necrotic crown and root tissues on 10% clarified V8-PARP (Jeffers and Martin 1986) yielded Phytophthora-like colonies. Three isolates were subcultured on V8 agar and chlamydospores and hyphal swellings were abundant in 2-week-old cultures. All three isolates produced abundant noncaducous and nonpapillate sporangia ranging from 33 to 54 μm × 20 to 39 μm (average 45.5 × 28.5 μm, n = 20) in soil water extract solution. Isolates did not produce oospores after 2 weeks on carrot agar at 20°C in the dark. Isolates had optimum vegetative growth at 30 oC and grew well at 35 oC. There was no growth at 5 and 40 oC. Genomic DNA was extracted from the mycelia of three isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. The internal transcribed spacer (ITS) region of rDNA, mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene, and beta-tubulin (β-tub) gene were amplified with primers ITS1/ITS4 (White et al., 1990), COXF4N/COXR4N and TUBUF2/TUBUR1 (Kroon et al., 2004) and the resulting 3 amplicons were sequenced (GenBank Accession No. OK438221, OK484426, and OK484427). A BLASTn search of 811-bp amplicon (OK438221) revealed 99% match (762/766) with ITS sequences MG865562 which was Phytophthora parsiana Ex-type CPHST BL 47 from Iran. BLAST analysis of the 867-bp amplicon (OK484427) showed 99% identity (866/867) with the COX 1 sequence of P. parsiana (KC733455) from Virginia. BLAST analysis of the 941-bp amplicon (OK484426) showed 99% identity (928/938) with the β-tub sequence of P. parsiana (AY659746). To fulfill Koch’s postulates, pathogenicity tests were conducted twice on 2-week-old ‘Az 2’ guayule seedlings grown in 10 plants per 1.9-liter pot filled with a steam-disinfested potting mix. Pots were placed in a plastic container and watered three times a week by flooding, to create waterlogged conditions. Plants were maintained in a greenhouse with 12 h day/12 h night (15-28 oC) and fertilized weekly with a 20-20-20 fertilizer at 1mg/ml. Fifty plants in 5 pots were challenged with a P. parsiana isolate by drenching each pot with 50 ml of a 1×105 zoospore/ml suspension. Fifty plants in 5 pots, serving as a control, received each 50 ml of distilled water. Symptoms of wilting, root rot, and plant death were observed 1 week afterward in inoculated plants, whereas control plants remained asymptomatic. P. parsiana was reisolated from necrotic roots of inoculated plants but not from control plants. To our knowledge, this is the first report of crown and root rot in guayule caused by P. parsiana in Arizona. P. parsiana is a species known for causing root rot on woody plants such as pistachio in California (Fichtner et al., 2016) and Iran (Mostowfizadeh-Ghalamfarsa et al., 2008). Arizona is home of desert woody guayule plant. P. parsiana may represent a significant barrier to commercialization of guayule for rubber in low desert areas of Arizona. The origin, distribution, and virulence of the pathogen on Arizona guayule is currently unknown. Disease resistance evaluation may help identify resistance in guayule germplasm that are useful in breeding for resistant cultivars.

First Report of bacterial speck caused by Pseudomonas syringae pv. tomato Race 1 affecting tomato in different Regions of Chile

In Chile, tomato is one of the most widely cultivated vegetables, with around 5,000 ha for fresh market and 8,000 ha for processing industry. During recent years, symptoms of bacterial speck caused by Pseudomonas syringae pv. tomato, have been observed more frequently in tomato plants in different regions of Chile. This pathogen was first identified in Chile in 1987 (Latorre & Lolas, 1988) and the presence of an apparent new variant was reported in 2004 (Besoain et al. 2004). To characterize the pathogen that was affecting this crop, samples of diseased tomato plants were taken in three regions of Chile. The samples were collected in 2016 in Northern Chile in Lluta Valley from the Arica y Parinacota Region, and in Central Chile, in 2014 in Limache from Valparaíso Region and in 2015 in Pichidegua from O´Higgins Region. Affected tomato plants exhibited dark brown to black lesions surrounded by yellow halos in the leaves, and dark brown to black lesions in the stems, pedicels, and peduncles. Plants tissues were macerated, and the suspension was spread on King’s B medium, resulting in fluorescent colonies visualized under 366 nm UV light. LOPAT tests results of three selected isolates from different Regions, were: levan production (+), oxidase reaction (-), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+) (Lelliot et al. 1966). Molecular identification was carried out by amplification and sequence analysis of housekeeping genes cts, encoding citrate synthase, gyrB, encoding DNA gyrase B, and rpoD, encoding sigma factor 70 (Hwang et al. 2005; Sarkar & Guttmann 2004) (GenBank Accessions No. OK001658-OK001666). BLAST analysis of cts and rpoD genes of the three isolates resulted in a match with a 100% identity (919 bp and 491 bp respectively) with Pseudomonas syringae pv. tomato strain B13-200 (GenBank: CP019871.1). BLAST analysis of gyrB gene of two isolates resulted in a match with a 100% identity (684 bp) and one isolate with 99.85% (683 bp) with Pseudomonas syringae pv. tomato strain B13-200. To identify the race 1, each strain was inoculated in five tomato plants cv. San Pedro, susceptible to both races of P. syringae pv. tomato, and cv. Rio Grande, resistant to race 0. The tomato plants were slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, including the reference strain DC3000 (race 0). Negative controls were sprayed with water. The plants inoculated with Chilean strains in both cv. San Pedro and cv. Rio Grande, showed symptoms of bacterial speck after 7 days. Plants inoculated with DC3000 strain showed symptoms only in cv. San Pedro, whereas control plants remained asymptomatic. Strains were re-isolated from symptomatic plants and identified by gene sequence analyses as Pseudomonas syryngae pv. tomato. This is the first report of Pseudomonas syryngae pv. tomato race 1 in Chile. Race 1 was previously reported in Canada (Lawton and MacNeill. 1986), in Italy (Buonaurio et al. 1996), in California (Arredondo and Davis 2000), in Portugal (Cruz et al. 2010), and in other states in the USA and countries in South America, Europe, Africa, and Australia, becoming the most commonly isolated race today (Cai et al 2011). These results will be the base for future studies of epidemiology, characterization, and virulence in order to explain the outbreak of this disease and the severity of symptoms observed.

Morphometric and genetic identification of a newly record pygmy seahorse Hippocampus denise in the Kepulauan Seribu reefs, Indonesia

The study was conducted to describe the seahorse species based on morphological and molecular characters. The pygmy seahorse in Panggang Island in Kepualuan Seribu was discovered in October 2011. The species was allegedly identified as Hippocampus denise (Family: Syngnathidae) described by Lourie and Randall which published in 2003. The high similarity is based on small morphometric, orange-like color and its association with sea fan Annella sp. Their habitat is fairly shallow at a depth between 13-24 meters compared with their sister species observed in Bali, Nusa Tenggara, and Sulawesi. The phylogenetic analysis constructed with several sequence data of Hippocampus spp. from Genbank shows that sample collected from Panggang Island is in the same clade with Hippocampus denise with 100% bootstrap value. BLAST analysis result also showed a high maximum similar identity (>99%) with the species Hippocampus denise. The seahorse specimen described in this study has a common typology of habitat with Hippocampus denise. This study shows that genetic analysis to determine the Hippocampus denise can be carried out to support species recognition, especially for cryptic species such as Hippocampus spp. There are variations in morphometric and habitat depth levels, indicating local adaptation of pygmy seahorses to the Kepulauan Seribu reefs.

Ship Blast Analysis

Naval vessels and Submarines structures in their fighting role are susceptible to explosion of torpedoes, mines, TNT etc. The damage inflicted by Contact explosion consists of direct shock wave damage to hull, whipping damage to keel and mechanical damage to onboard equipment and associated systems. The order to design a shock resistant structure, it is important to simulate these structures and loads and then subsequently analyze the same to predict the response (as performing experiments would be expensive). The TNT (Trinitrotoluene) explosion analysis of large structures like ships could be considered as one of the most complicated numerical analysis. Loads can be calculated by using published empirical formulas, which are complicated if calculated the large structure. By using of the FE Software ANSYS, backed up with in the developed software, for Explosion analysis of structures.

Computationally predicted virus responsive miRNAs in banana (Musa AAB)

The bioinformatics tool NovoMIR was used to predict miRNAs in Musa acuminata genome. NovoMIR predicted 85 pre-miRNAs from the 11 chromosomes of Musa acuminata DH Pahang and BLAST analysis of the predicted pre-miRNAs against annotated miRNAs of miRBase identified 52 mature miRNAs belonging to 38 different families. psRNATarget server identified 124 protein-coding sequences as potential targets for 40 mature miRNAs. Based on the role of the target genes in biological processes related to biotic stress, five miRNAs were selected for analyzing their response to Banana bract mosaic virus (BBrMV) infection. Three-month-old in vitro raised banana plants of var. Nendran (Musa AAB) infected with BBrMV showed the presence of all the five selected miRNAs in both healthy and BBrMV infected plants. Expression analysis using RT-qPCR showed changes in the expression of miR-3900-5p, miR-2172-5p, miR-6928-5p and miR-971-5p and their targets during BBrMV infection.

Identification of 16SrXXX-A Phytoplasma Associated with Salt cedar (Tamarix chinensis Lour.) witches'-broom (SCWB) in Southern Xinjiang of China

Salt cedar is an ornamental shrub/moderate tree species native to Asia and East Europe, and grows in salt-alkali soil, desert and other dry areas, which plays an important role in wind prevention and sand fixation as well as maintaining ecological balance. Salt cedar witches’-broom (SCWB), which was extremely pernicious to Salt cedar. It was first observed and reported in Xi’an, China in 2005 (Zhao et al.2005). Witches' broom symptoms were observed on 20 out of 150 (13.3%) salt plants surveyed from the Alar region and 10 out of 86 (11.6%) plants from the Akesu region in southern of Xinjiang in May 2020. The damaged plants compared with asymptomatic plants (Fig.1A), the major symptoms included branches clustered, intersegment shorten and coarsen, giving rise to the formation of clusters (Fig.1B). Total plant DNA was extracted from phloem tissues with asymptomatic symptoms and phloem tissues with witches'-broom symptoms by a CTAB-based DNA extraction method (Green et al.1999). The 16S rRNA gene and the phytoplasma universal primers P1/P7 and rpF1/rpR1 of the rp (ribosomal protein) gene were used for Polymerase chain reaction (PCR) amplification by using the extracted plant total DNA as the template. The PCR product was used as the template and the R16F2n/R16R2 prmer was used for nested PCR amplification of the 16S rRNA gene after the amplification was completed. The results show that no product was obtained in asymptomatic plants. When DNA samples from witches’-broom symptomatic plants were used as templates, fragments with lengths 1219 bp and 1174 bp, corresponding to 16S rRNA gene and rp gene, were obtained. 16S rRNA gene was sequenced and deposited in GenBank under accession number MW447513. BLAST analysis revealed that the partial 16S rRNA sequence of the phytoplasma associated with P. aphylla witches’ broom showed highest sequence identity (99.67%) to salt cedar witches’ broom phytoplasma, ‘Candidatus Phytoplasma tamaricis’ (Accession Number: FJ432664). Phylogenetic and molecular evolutionary analyses were conducted using MEGA-X (Kumar et al., 2018). Results showed taht the SCWB and 16S rXXX group’s‘Candidatus Phytoplasma tamaricis’, (GenBank accession: FJ432664) have the highest affinity (Fig.2A). A virtual restriction fragment length polymorphism(RFLP) was done to determinethe subgroup ( Zhao et al. 2009). The 16S rDNA sequence from the Tamarix chinensis plant showed 99.3% similarity with that of the “Candidatus Phytoplasma tamaricis” reference strain (GenBank accession: FJ432664), suggesting that the phytoplasma in this study belongs to “Candidatus Phytoplasma tamaricis”-related strain. Therefore, it can be stated that SCWB belongs to the 16S rXXX group. The partial rp sequences only shared 84.74% sequence similarity with that of ‘Candidatus Phytoplasma prunorum’ (MG383523) of Apple proliferation group, a known subgroup 16S rX. Blast analysis based on the partial rp sequences showed that it shares less than 90% similarity with that of any known phytoplasma (Fig 2B), we suspect that this is due to a lack of sequenced rp gene sequences for the 16S rXXX group. To our knowledge, this is the first report of Salt Cedar Witches' Broom phytoplasma in Xinjiang province, China. As a consequence, we guess the SCWB phytoplasma rp gene belongs to 16S rXXX-rp group, which is also the first report about the 16SrXXX-rp group. Because SCWB1 is the only strain in the 16S rXXX group, and it is the representative strain of the 16S rXXX-A subgroup (Zhao et al. 2009). So, the SCWB disease we found in southern Xinjiang belongs to the 16S rXXX-A subgroup.

Building of an Internal Transcribed Spacer (ITS) Gene Dataset to Support the Italian Health Service in Mushroom Identification

This study aims at building an ITS gene dataset to support the Italian Health Service in mushroom identification. The target species were selected among those mostly involved in regional (Tuscany) poisoning cases. For each target species, all the ITS sequences already deposited in GenBank and BOLD databases were retrieved and accurately assessed for quality and reliability by a systematic filtering process. Wild specimens of target species were also collected to produce reference ITS sequences. These were used partly to set up and partly to validate the dataset by BLAST analysis. Overall, 7270 sequences were found in the two databases. After filtering, 1293 sequences (17.8%) were discarded, with a final retrieval of 5977 sequences. Ninety-seven ITS reference sequences were obtained from 76 collected mushroom specimens: 15 of them, obtained from 10 species with no sequences available after the filtering, were used to build the dataset, with a final taxonomic coverage of 96.7%. The other 82 sequences (66 species) were used for the dataset validation. In most of the cases (n = 71; 86.6%) they matched with identity values ≥ 97–100% with the corresponding species. The dataset was able to identify the species involved in regional poisoning incidents. As some of these species are also involved in poisonings at the national level, the dataset may be used for supporting the National Health Service throughout the Italian territory. Moreover, it can support the official control activities aimed at detecting frauds in commercial mushroom-based products and safeguarding consumers.

Prevalence and molecular identification of Nematodirus helvetianus in camels in Iraq

Background and Aim: Camels from the central part of Iraq are infected with multiple parasitic diseases that have an economic impact by decreasing meat and milk production. This study aimed to evaluate Nematodirus spp. in camels (Camelus dromedarius). Materials and Methods: The study animals consisted of camels slaughtered in the central area of Iraq at the Al-Najaf slaughterhouse. All ages and sexes of camels were examined. Worms were recovered and identified microscopically. For molecular characterization, two Iraqi Nematodirus spp. partial ribosomal genes (ITS1 and ITS2) were sequenced and submitted to the NCBI database. Results: Of 160 camels tested, 29 were infected with Nematodirus spp. (18.13%). Twenty-one nematodes containing the Nematodirus genes were identified in the small intestines of naturally infected camels. BLAST analysis revealed 88.1% sequence similarity with that of Nematodirus helvetianus isolated in China and 87.2% similarity with N. helvetianus isolated in the United States. Conclusion: The prevalence of N. helvetianus warrants the use of anti-helminthic drugs for these animals and a rationale for future control strategies to prevent the transmission of this infection to other livestock.

Isolation and identification of peste des petits ruminants virus from goats in Egyptian governorates

Background and Aim: The peste des petits ruminants (PPR) is a highly contagious disease of small ruminants which negatively affects animal production and the socioeconomic status of farmers. Peste des petits ruminants virus (PPRV) encodes eight proteins, with the viral fusion protein (F) playing a role in virus virulence and stimulating an effective protective immune response. This study aimed to isolate and complete the identification of PPRV circulating in goats in different Egyptian governorates and perform molecular characterization of the PPRV F gene. Materials and Methods: Samples were collected from unvaccinated animals with clinical signs suggestive of PPR. A total of 256 sera were tested for the detection of PPRV antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA) kit, while 214 samples of blood buffy coat preparation, animal swabs (nasal, ocular, and saliva), and fecal and tissue samples were tested for the detection of the PPRV antigen using an antigen-capture ELISA kit. Molecular diagnosis, gene cloning, blast analysis, and phylogenetic analysis were performed for the molecular characterization of PPRV. Results: The seroprevalence results of PPRV antibodies in the tested sera showed a total of 67.9% positive samples. The rates of PPR antigen recorded by the antigen-capture ELISA in the swabs (nasal and ocular) and tissue samples were 44.3%, 46.8%, and 43.5%, respectively, with saliva swabs having the highest rate of PPRV positivity (76.4%) and fecal samples having the lowest (33.3%). Molecular characterization of the PPRV Vero cell culture revealed that the circulating PPRV strain belongs to the IV lineage. Blast analysis of the PPRV F gene showed 96.7% identity with the PPRV strain Egypt-2014 fusion protein (F) gene, KT006589.1, differing by 43 single-nucleotide polymorphisms. Conclusion: The results of this study indicate that the emerging PPRV belongs to the IV lineage among small ruminant animals. The findings also indicate the need for an innovative strategy to control and eliminate this disease based on a regularly administered and effective vaccine, a test to distinguish between infected and vaccinated animals, and the need for further study on the protein structure and PPRV F gene expression, which should help us to understand the molecular evolution of the virus and control and eliminate PPR disease.