Meloidogyne aegracyperi n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing yellow and purple nutsedge in New Mexico.

Meloidogyne aegracyperi n. sp. is described from roots of purple nutsedge in southern New Mexico, USA. Mature females are small (310-460 µm), pearly white, with their egg masses completely contained inside root galls. The neck is often at a 90 to 130° angle to the protruding posterior end with the perineal pattern. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet is relatively long (4.0-6.1 µm), and the excretory pore is level with the base of the stylet. The anterior portion of the rounded lumen lining of the metacorpus contains 3 to 10 small vesicles. The perineal pattern has a rounded dorsal arch with a tail terminal area that is smooth or marked with rope-like striae. Only two males were found. The body twists 90° throughout its length. The DGO to the base of the stylet is long (3.0-3.3) µm. The cephalic framework of the second-stage juvenile is weak, and the stylet is short (10.1-11.8 µm). The DGO to the base of the stylet is long (3-5 µm). The tail is very long (64-89 µm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Eggs are typical for the genus and vary in length (85.2-99.8 µm) and width (37.1-48.1 µm), having a L/W ratio of (2.1-2.6). Maximum likelihood phylogenetic analyses of the different molecular loci (partial 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA gene fragments and partial Hsp90 gene) placed this nematode on an independent branch in between M. graminicola and M. naasi and a cluster of species containing M. chitwoodi. M. fallax, and M. minor. Greenhouse tests showed that yellow and purple nutsedge were the best hosts, but perennial ryegrass, wheat, bentgrass, and barley were also hosts.

New Mexico and the southwestern US are affected by a unique population of tomato spotted wilt virus (TSWV) strains.

Tomato spotted wilt virus (TSWV) is an important pathogen of many ornamental, greenhouse and agronomic crops worldwide. TSWV also causes sporadic problems in a number of crops in New Mexico (NM). Nucleocapsid gene sequences obtained from six different crop species across the state over four different years were used to characterize the NM TSWV population. This analysis shows that NM is affected by a unique TSWV population that is part of larger independent population present in the southwestern US. This population likely arose due to geographic isolation and is related to other TSWV populations from the US, Spain, and Italy.

First Report of Xylella fastidiosa in Peach in New Mexico.

Xylella fastidiosa is a gram-negative bacterium that causes disease in a wide variety of plants such as grapes, citrus trees, oleanders, and elm and coffee trees. This bacterium is xylem limited and causes disease symptoms such as leaf scorch, stunting of plant growth, branch dieback, and fruit loss. The presence of X. fastidiosa was previously reported in New Mexico where it was found to be infecting chitalpa plants and grapevines (3). In the summer of 2010, peach (Prunus persica (L.) Batsch) trees from two locations in northern New Mexico exhibited leaf deformity and stunting, dark green venation, slight mottling, and branch dieback. Preliminary viral diagnostic screening was performed by Agdia (Elkhart, IN) on one symptomatic tree and it was negative for all viruses tested. Three trees from two different orchards tested positive for X. fastidiosa by ELISA and PCR analysis using X. fastidiosa-specific primer sets HL (1) and RST (2). Bacterial colonies were also cultured from these samples onto periwinkle wilt media. Eight colonies obtained from these three plants tested PCR positive using the X. fastidiosa-specific primers. The 16S ribosomal and 16S-23S rRNA internal transcribed spacer (ITS) region (557 nucleotides) (GenBank Accession No. HQ292776) along with the gyrase region (400 nucleotides) (GenBank Accession No. HQ292777) was amplified from the peach total DNA samples and the bacterial colonies. Sequencing analysis of these regions indicate that the X. fastidiosa found in peach is 100% similar to other X. fastidiosa multiplex isolates including isolates from peach, pecan, sycamore, and plum trees and 99% similar to the X. fastidiosa isolates previously found in New Mexico. Further analysis of the 16S ribosomal and 16S-23S rRNA ITS sequences with maximum likelihood phylogenetic analysis using Paup also groups the peach isolates into the X. fastidiosa multiplex subspecies. The gyrase sequence could not be used to differentiate the peach isolates into a subspecies grouping because of the lack of variability within the sequence. This X. fastidiosa multiplex subspecies could possibly be a threat to the New Mexico pecan industry since pecan infecting X. fastidiosa isolates belong to the same bacterial subspecies. It is not known if X. fastidiosa subspecies multiplex isolates from peach are capable of infecting pecans but they are closely genetically related. It is interesting to note that the isolates from peach are different than previously described X. fastidiosa isolates in New Mexico that were infecting chitalpa and grapes (3). X. fastidiosa has previously been described in peach; the disease is called "phony peach". The peach trees exhibited stunting and shortened internodes as reported for "phony peach". They also exhibited slight mottling and branch dieback that may be due to the environment in New Mexico or perhaps they are also exhibiting mineral deficiency symptoms in association with the X. fastidiosa disease. To our knowledge, this is the first report of X. fastidiosa in peach in New Mexico. References: (1) M. H. Francis et al. Eur. J. Plant Pathol. 115:203, 2006. (2) G. V. Minsavage et al. Phytopathology 84:456, 1994. (3) J. J. Randall et al. Appl. Environ. Microbiol. 75:5631, 2009.

Brote Grande, a New Phytoplasma-Associated Disease of Chile Peppers.

Chile is one of the most important crops in New Mexico, contributing both to the agricultural economy and cultural identity of the state. Chile producers in New Mexico and Arizona have reported a disorder of unknown etiology that has increased in frequency for the past several years. Affected plants have a bushy appearance, develop overly large green calyces instead of normal flowers, and fail to set fruit. This characteristic phyllody is similar to symptoms associated with other phytoplasma-caused diseases, such as tomato big bud, and has led chile producers to refer to the disorder as "brote grande", which is Spanish for "big bud". PCR analysis using the phytoplasma-specific primer pairs P1/Tint and P1/P7 (4) produced amplicons of the expected size (~1.6 kb) from symptomatic but not healthy samples. Direct sequencing of the P1/P7 PCR amplicons determined that they contained the expected 16S rRNA and internal transcribed spacer (ITS) sequences and included the tRNAIle typically found in phytoplasma ITS regions. BLAST analysis of the brote grande sequence (GenBank Accession No. FJ525437) indicated it is most closely related (99% identity) to sequences reported for previously characterized 16Sr group VI phytoplasmas, such as 'Candidatus Phytoplasma trifolii' (Accession No. AY390261) and the Vinca virescence (Accession No. AY500817) phytoplasma. 'Candidatus phytoplasma trifolii' is synonymous with beet leafhopper virescence, which was reported as a cause of tomato big bud in California during the mid 1990s (3). The brote grande phytoplasma was less related to other phytoplasmas known to affect peppers such as the 16Sr group XII stolbur of pepper phytoplasma (Accession No. AF248959) and newly described 16Sr group I phytoplasmas described in peppers in Cuba (Accession No. DQ286947) and Mexico (Accession No. DQ092321) (1,2). The brote grande phytoplasma is also distinct from other phytoplasmas, such as potato purple top and tomato little leaf that are common in Mexico, affecting solanaceous crops in the region (2). Although the disease frequency never exceeded 5% in any given field, plants displaying brote grande symptoms were observed in the majority of chile pepper fields examined from July to September of 2008. The presence of the brote grande associated phytoplasma was confirmed by PCR and sequence analysis of symptomatic plants from 10 different fields ranging from Las Cruces, NM to Tucson, AZ, indicating that brote grande disease is widespread across the major chile-producing areas of the Desert Southwest. The brote grande phytoplasma sequence was the only phytoplasma sequence detected in any of the symptomatic chile samples. Taken together, the etiology, PCR, and DNA sequence results all indicate that brote grande of chile is a new disease of chile peppers associated with infection by a novel 16Sr group VI phytoplasma and that this disease is distributed across the major chile-producing areas of the Desert Southwest. References: (1) Y. Arocha. Plant Pathol. 56:345, 2007. (2) M. E. Santos-Cervantes et al. Plant Dis. 92:1007, 2008. (3) M. E. Shaw et al. Plant Dis. 77:290, 1993. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

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 Pierce's 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.

Establishing guidelines to improve identification of fire ants Solenopsis xyloni and Solenopsis invicta.

As red imported fire ant, Solenopsis invicta Buren, continues to expand its range into the southwestern United States, it can be easily confused with the native southern fire ant, Solenopsis xyloni McCook. Variability in the morphological characteristics commonly used to differentiate these ant species was quantified by examining the length of the clypeal tooth, striations of the mesopleuron, length of antennal scape, area of the petiolar process, number and size of mandibular teeth, and color by using both scanning electron and light microscopy. Given enough samples, the average values of each of these characteristics is different between the two species; however, significant morphological variability occurs in both S. xyloni and S. invicta populations, creating an area of overlap where either of the two species could exhibit similar characteristics. Better differentiation of these two species is achieved using a combination of characteristics, but morphological techniques are not dependable unless numerous ants from each population are analyzed by a taxonomist familiar with Solenopsis. For situations requiring a more accurate identification, such as before quarantining a county or a portion of a county, a molecular technique using mitochondrial DNA and polymerase chain reaction techniques was developed.

Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps.

Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sf-caspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD downward arrowG between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD downward arrow(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD downward arrowG processing of pro-Sf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD downward arrowG processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.

trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants.

ABSTRACT Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.

Mutational analysis of the putative nicking motif in the replication-associated protein (AC1) of bean golden mosaic geminivirus.

Geminiviruses are circular single-stranded DNA viruses that replicate by a rolling circle mechanism involving the viral-encoded AC1 protein. DNA nicking is necessary both for initiating replication of the covalently closed double-stranded DNA templates and for releasing unit-length monomers. The effects of mutations in a putative nicking motif (K101 A Y I D K106; E. V. Koonin and T. V. Ilyina, J. Gen. Virol. 73:2763-2766, 1992) of the AC1-derived protein for bean golden mosaic geminivirus isolate GA (BGMV-GA) were studied. The amino acids equivalent to Y103 and K106 of BGMV-GA are invariant in all whitefly-transmitted geminiviruses. Phaseolus vulgaris plant infectivity assays showed that the mutants K101-->H, K101-->A, and D105-->T produced symptoms, but mutants Y103-->A, Y103-->F, K106-->R, and K106-->H did not. A mutant with a stop codon in the N terminus of the AC4 open reading frame (ORF) produced the same symptoms as the wild-type BGMV-GA. Only those that were infectious replicated in NT-1 tobacco suspension cells. These results indicate that the Y103 and K106 residues are essential for replication, and that this putative DNA-nicking motif of the AC1 ORF may be functional in the rolling circle mechanism of replication for geminiviruses. The potential role of these mutants in the design of antiviral strategies is discussed.

Mutational analysis of a putative NTP-binding domain in the replication-associated protein (AC1) of bean golden mosaic geminivirus.

Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA geminivirus with a bipartite genome. AC1 is the only ORF required for geminiviral replication. A putative NTP-binding motif, EGX4GKTX32DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263). We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies. Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from K228 to H or D262 to R within the putative NTP-binding site. A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain. Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from 1190 to R. Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication. The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed.