Jennifer J. Randall

Genetic Analysis of a Novel Xylella fastidiosa Subspecies Found in the Southwestern United States

ABSTRACT Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.

Identification of a signal peptide for oryzacystatin-I.

Abstract. A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.

Complete Genome and Plasmid Sequences for Rhodococcus fascians D188 and Draft Sequences for Rhodococcus Isolates PBTS 1 and PBTS 2.

ABSTRACT Rhodococcus fascians, a phytopathogen that alters plant development, inflicts significant losses in plant production around the world. We report here the complete genome sequence of R. fascians D188, a well-characterized model isolate, and Rhodococcus species PBTS (pistachio bushy top syndrome) 1 and 2, which were shown to be responsible for a disease outbreak in pistachios.

BABA and Phytophthora nicotianae Induce Resistance to Phytophthora capsici in Chile Pepper (Capsicum annuum).

Induced resistance in plants is a systemic response to certain microorganisms or chemicals that enhances basal defense responses during subsequent plant infection by pathogens. Inoculation of chile pepper with zoospores of non-host Phytophthora nicotianae or the chemical elicitor beta-aminobutyric acid (BABA) significantly inhibited foliar blight caused by Phytophthora capsici. Tissue extract analyses by GC/MS identified conserved change in certain metabolite concentrations following P. nicotianae or BABA treatment. Induced chile pepper plants had reduced concentrations of sucrose and TCA cycle intermediates and increased concentrations of specific hexose-phosphates, hexose-disaccharides and amino acids. Galactose, which increased significantly in induced chile pepper plants, was shown to inhibit growth of P. capsici in a plate assay.

First Report of Phytophthora nicotianae on Bulb Onion in the United States

Phytophthora nicotianae (synonym P. parasitica) Breda de Haan was isolated from recently harvested onion bulbs (Allium cepa) in cold storage from a commercial field in southern New Mexico. Deteriorating, water-soaked tissue from the center of four bulbs was plated onto water agar and incubated at room temperature. After 72 h, cultures of Phytophthora (identified by the presence of coenocytic hyphae and papillate sporangia) were isolated and transferred to V8 agar amended with ampicillin (250 mg/liter), rifampicin (10 mg/liter), and pimaricin (0.2% wt/vol). Isolates were identified as P. nicotianae based on morphological characteristics and DNA analysis. Sporangia were sharply papilliate, noncaducous, and ovoid to spherical. The average sporangium size was 45.9 × 39.9 μm with a length-to-width ratio of 1.15. Clamydospores, both terminal and intercalary, were spherical to ovoid and averaged 37.2 × 35.2 μm (2). PCR from whole-cell extracts was performed on four cultured isolates from the infected onion tissue using previously described primers ITS4 and ITS6, which amplify the 5.8S rDNA and ITS1 and ITS2 internal transcribed spacers (1,4). A band of approximately 890 bp was amplified and directly sequenced (GenBank Accession No. HQ398876). A BLAST search of the NCBI total nucleotide collection revealed a 100% similarity to multiple P. nicotianae isolates previously sequenced (1). To confirm the pathogenicity of the isolates, onion seedlings were inoculated with 25 ml of P. nicotionae zoospore solution (15,000 zoospores/ml). Necrosis of leaf tissue and seedling death was observed 5 days postinoculation. P. nicotianae was reisolated from the infected onion seedlings and the ITS region was sequenced to confirm its identity. P. nicotianae was previously reported in bulb onion from Australia, Taiwan (Formosa), and Zimbabwe (Rhodesia) (2). P. nicotianae was reported on bunching onions (A. fistulosum) in Hawaii in 1989 (3). Onions are an important crop in New Mexico with a total production value of 47 million dollars in 2008 (NM Agriculture Statistics 2008). This discovery of a potentially significant postharvest disease poses a threat to the onion industry in New Mexico. To our knowledge, this is the first report of P. nicotianae in bulb onion in the United States and the first report of P. nicotianae in New Mexico on any crop. References: (1) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (2) D. C. Erwin and O. K. Ribeiro. Page 56 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St Paul, MN, 1996. (3) R. D. Raabe et al. Information Text Series No. 22. University of Hawaii. Hawaii Inst. Trop. Agric. Human Resources, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Xylella fastidiosa Detected in New Mexico in Chitalpa, a Common Landscape Ornamental Plant

Different strains of Xylella fastidiosa cause a variety of significant disease problems in agricultural and ornamental plants, including Pierces disease in grapes, oleander leaf scorch, pecan bacterial leaf scorch, and alfalfa dwarf disease. X. fastidiosa has never been reported in New Mexico but is known to exist in surrounding states (California, Arizona, and Texas). During the summer of 2006, several chitalpa (Chitalpa tashkinensis) hybrid trees with leaf scorch symptoms and branch die back were observed in Las Cruces, NM and they tested positive for X. fastidiosa by ELISA. Additional samples from these plants and others were analyzed by ELISA, PCR (2), and cultured on XfD2 medium (1). Known positive and negative oleander samples from Arizona were included as controls. Fifteen of thirty tested chitalpa were PCR and ELISA positive, indicating that they were infected with X. fastidiosa. Bacterial colonies that were PCR positive were also recovered from 10 of the XF positive samples that were plated. DNA sequences of PCR products amplified from chitalpa and isolated bacterial colonies (GenBank Accession Nos. EF109936 and EF109937) were identical to each other, 97% similar to X. fastidiosa strain JB-USNA, and 96% similar to the Temecula 1 strain. Independent ELISA testing (Barry Hill, California Department Food and Agriculture, Sacramento, CA) confirmed our ELISA and PCR results. On the basis of these results, we conclude that X. fastidiosa is present in New Mexico and that the common landscape ornamental chitalpa is a host for X. fastidiosa. Additional work is required to determine if X. fastidiosa is pathogenic to chitalpa and to examine the relevance of this potential X. fastidiosa reservoir to agricultural production in New Mexico and other areas where chitalpa is grown. References: (1) R. P. P. Almeida et al. Curr. Microbiol. 48:368, 2004. (2) M. R. Pooler et al. Lett. Appl. Microbiol. 25:123, 1997.

Comment on “Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management”

We would like to address a number of concerns regarding this paper (Savory et al., 2017)

Phytophthora Species in Rivers and Streams of the Southwestern United States

ABSTRACT Phytophthora species were isolated from rivers and streams in the southwestern United States by leaf baiting and identified by sequence analysis of internal transcribed spacer (ITS) ribosomal DNA (rDNA). The major waterways examined included the Rio Grande River, Gila River, Colorado River, and San Juan River. The most prevalent species identified in rivers and streams were Phytophthora lacustris and P. riparia, both members of Phytophthora ITS clade 6. P. gonapodyides, P. cinnamomi, and an uncharacterized Phytophthora species in clade 9 were also recovered. In addition, six isolates recovered from the Rio Grande River were shown to be hybrids of P. lacustris × P. riparia. Pathogenicity assays using P. riparia and P. lacustris failed to produce any disease symptoms on commonly grown crops in the southwestern United States. Inoculation of Capsicum annuum with P. riparia was shown to inhibit disease symptom development when subsequently challenged with P. capsici, a pathogenic Phytophthora species. IMPORTANCE Many Phytophthora species are significant plant pathogens causing disease on a large variety of crops worldwide. Closer examinations of streams, rivers, and forest soils have also identified numerous Phytophthora species that do not appear to be phytopathogens and likely act as early saprophytes in aquatic and saturated environments. To date, the Phytophthora species composition in rivers and streams of the southwestern United States has not been evaluated. This article details a study to determine the identity and prevalence of Phytophthora species in rivers and streams located in New Mexico, Arizona, Colorado, Utah, and Texas. Isolated species were evaluated for pathogenicity on crop plants and for their potential to act as biological control agents.

First report of anthracnose of sunflower sprouts caused by Colletotrichum acutatum in New Mexico.

In December 2011, edible sunflower sprouts (Helianthus annus) of two different commercially grown cultivars (Sungrown and Tiensvold) exhibiting stem and cotyledon lesions were submitted to the New Mexico State University Plant Clinic for disease diagnosis. The sample originated from an organic farm in Santa Fe County where the grower utilizes a small indoor growing facility. Stem lesions were elongate, reddish brown, and often constricted, resulting in stem girdling. Lesions on the cotyledons were dark brown with tan centers and round to irregular in shape. In some cases, the entire cotyledon was blighted. Fungal hyphae were observed on some lesions using a dissecting microscope. Colletotrichum acutatum was isolated from stem and cotyledon lesions when symptomatic tissue was plated on water agar. Conidia were fusiform ranging from 6.4 to 18.4 μm long and 2.1 to 5.1 μm wide and averaged 11.9 μm × 3.4 μm. Spores were measured from cream-colored colonies produced on acidified potato dextrose agar. PCR amplification and sequence analysis of 5.8S ribosomal DNA and internal transcribed spacers I and II was performed using primers ITS4 and ITS6 (2). An amplification product of approximately 600 base pairs in size was directly sequenced (GenBank Accession No. JX444690). A BLAST search of the NCBI total nucleotide collection revealed a 99% identity to multiple C. acutatum (syn: C. simmondsii) isolates. Four isolates were identified as C. acutatum based on morphological characteristics and DNA analysis. Kochs postulates were performed using four isolates of the pathogen and the two commercial sunflower cultivars (Sungrown and Tiensvold) originally submitted for disease analysis. Sunflower seeds were imbibed in distilled water for 24 h then sewn into peat plugs. Prior to seed germination, 5 ml of a C. acutatum spore solution (1 × 106/ml) from each isolate was applied to five peat plugs using an atomizer. Control plants were inoculated with distilled water and otherwise treated identically. Both sunflower cultivars were inoculated with each isolate of the pathogen and the test was replicated twice. The sewn peat plugs were incubated for 5 days at 21°C and 50% relative humidity. Symptoms similar to the original samples were present on 100% of the sprouts after 5 days. PCR and sequence analysis performed on cultures obtained from lesions showed a 100% match to the original New Mexico isolates fulfilling Kochs postulates. In an indoor organic facility, such as the one in NM, this disease has the potential to be very difficult to manage and the potential to infect a high percentage of the crop resulting in significant economic losses. To our knowledge, this is the second report of C. acutatum on sunflower sprouts in the United States (1) and the first report in New Mexico. References: (1) S. T. Koike et al. Plant Dis. 93:1351, 2009. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.