Construction of an Infectious Clone of the Badnavirus Cacao Swollen Shoot Ghana M Virus and Infectivity by Gene Gun- and Agrobacterium-Mediated Inoculation

Cacao swollen shoot disease (CSSD) is a damaging disease of Theobroma cacao L. associated with infection by a group of poorly characterized badnaviral species. To establish causality and characterize the symptomatology associated with infection by the badnavirus cacao swollen shoot Ghana M virus (CSSGMV), an infectious clone (1.3-mer) was constructed and used to inoculated cacao “Amelonado” seedlings by biolistic inoculation (BI; n = 18) and agroinoculation (AI; n = 15). Newly expanded leaves of BI (10/18) and AI (12/15) plants developed foliar mosaic and curling symptoms 30-days post inoculation (dpi), with chlorotic mottling and necrotic crinkling being evident by 90 dpi. By 120 dpi, three of 15 AI plants exhibited characteristic stem-swelling. Viral infection was verified by PCR-amplification and sequencing of a 1068 bp fragment of the CSSGMV ORF3 from newly expanding leaves 60 dpi. The PCR results indicated that 14 of 18 and 15 of 15 BI and AI plants, respectively, were systemically infected. The complete CSSGMV genome sequence was determined, by Illumina sequencing, from representative AI and BI plants and shared >99.5% pairwise nucleotide identity with CSSGMV-Nig9 (GenBank Accession No. MH785299). Based on the development of characteristic CSSD symptoms and recovery of partial and complete genome sequences of CSSGMV-Nig9 from systemically infected cacao plants, Koch's postulates have been fulfilled.

Identification and Characterization of a Novel Emaravirus From Grapevine Showing Chlorotic Mottling Symptoms

A novel negative-sense, single-stranded (ss) RNA virus was identified in a “Shennong Jinhuanghou” (SJ) grapevine showing severe chlorotic mottling symptoms by integrating high-throughput sequencing (HTS) and conventional Sanger sequencing of reverse transcription polymerase chain reaction (RT-PCR) products. The virus was provisionally named as “grapevine emaravirus A” (GEVA). GEVA had a genome comprising five genomic RNA segments, each containing a single open reading frame on the viral complementary strand and two untranslated regions with complementary 13- nt stretches at the 5′ and 3′ terminal ends. RNA1 (7,090 nt), RNA2 (2,097 nt), RNA3 (1,615 nt), and RNA4 (1,640 nt) encoded putative proteins P1–P4 that, based on their conserved motifs, were identified as the RNA-dependent RNA polymerase, glycoprotein, nucleocapsid protein, and movement protein, respectively. However, the functional role of protein P5 encoded by RNA5 (1,308 nt) could not be determined. Phylogenetic trees constructed based on amino acids of P1 to P4, allocated GEVA in clade I, together with other species-related emaraviruses. These data support the proposal that GEVA is a representative member of a novel species in the genus Emaravirus of the family Fimoviridae. Moreover, when GEVA was graft-transmitted to SJ and “Beta” grapevines, all grafted plants showed the same symptoms, similar to those observed in the source of the inoculum. This is the first report to our knowledge of an emaravirus infecting grapevine and its possible association with chlorotic mottling symptoms.

Development of a Full-Length Infectious cDNA Clone of the Grapevine Berry Inner Necrosis Virus

Grapevine berry inner necrosis virus (GINV) belongs to the genus Trichovirus in the family Betaflexiviridae. The GINV isolate LN_BETA_RS was obtained from a “Beta” grapevine (Vitis riparia × Vitis labrusca) exhibiting chlorotic mottling and ring spot in Xingcheng, Liaoning Province, China. To verify the correlation between GINV and grapevine chlorotic mottling and ring spot disease, we constructed an infectious cDNA clone of GINV isolate LN_BETA_RS using the seamless assembly approach. Applied treatments of agroinfiltration infectious cDNA confirmed systemic GINV infection of the Nicotianaoccidentalis 37B by reverse transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy, exhibiting chlorotic mottling symptoms on leaves. Infectious cDNA was also transmitted to new healthy N. occidentalis plants through rub-inoculation. Moreover, the cDNA clone was agroinfiltrated into “Beta” and “Thompson Seedless” grapevine plantlets, and the inoculated grapevines exhibited leaf chlorotic mottling and ringspot during the two years of observation. GINV-inoculated “Beta” grapevines had serious leaf chlorotic mottling and ringspot symptoms on the whole plant, while relatively few symptoms were observed on the leaves of agroinoculated “Thompson Seedless” grapevines in early spring and only weak ring spot gradually appeared later in the top young leaves. Our experiments fulfilled Koch’s postulates and revealed the causative role of GINV in grapevine chlorotic mottling and ring spot disease.

Yellowing Disease in Zucchini Squash Produced by Mixed Infections of Cucurbit yellow stunting disorder virus and Cucumber vein yellowing virus

Zucchini squash is host to Cucurbit yellow stunting disorder virus (CYSDV), a member of the genus Crinivirus, and Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, both transmitted by the whitefly Bemisia tabaci. Field observations suggest the appearance of new symptoms observed on leaves of zucchini squash crops when both viruses were present. When infected during controlled experiments with CYSDV only, zucchini plants showed no obvious symptoms and the virus titer decreased between 15 and 45 days postinoculation (dpi), after which it was no longer detected. CVYV caused inconspicuous symptoms restricted to vein clearing on some of the apical leaves and the virus accumulated progressively between 15 and 60 dpi. Similar accumulations of virus followed single inoculations with the potyvirus Zucchini yellow mosaic virus (ZYMV) and plants showed severe stunting, leaf deformation, and mosaic yellowing. However, in mixed infections with CYSDV and CVYV, intermediate leaves showed chlorotic mottling which evolved later to rolling, brittleness, and complete yellowing of the leaf lamina, with exception of the veins. No consistent alteration of CVYV accumulation was detected but the amounts of CYSDV increased ≈100-fold and remained detectable at 60 dpi. Such synergistic effects on the titer of the crinivirus and symptom expression were not observed when co-infected with ZYMV.

Identification and Properties of a Carlavirus Causing Chlorotic Mottle of Florists' Hydrangea (H. macrophylla) in the United States

A previously uncharacterized virus with flexuous filamentous particles 660 nm in length was identified in the United States in florists' hydrangea (Hydrangea macrophylla), in which it caused chlorotic mottling, leaf deformation, and discoloration. The virus, tentatively named Hydrangea chlorotic mottle virus (HdCMV), was transmitted readily by mechanical inoculation and by Myzus persicae, but infected only H. macrophylla. The amino acid sequence of a 1.7-kb amplicon comprising the 3′ terminus of the HdCMV genome contained one partial and three complete putative open reading frames (ORFs) most similar in size, arrangement, and sequence to the homologous regions of the genomes of known carlaviruses. Based on virion morphology, genome properties, and current criteria for species demarcation, it was concluded that HdCMV represented a new species in the genus Carlavirus. Hydrangea ringspot virus (HdRSV, genus Potexvirus) occurred in mixed infections with HdCMV, but the two viruses could be distinguished readily by serological tests.

First Report of Xylella fastidiosa in Avocado in Costa Rica

Since the late 1990s, chlorotic mottling, marginal scorch, deformation of leaves, defoliation, shortening of internodes, and branch dieback have been observed in avocado trees (Persea americana Mill.) in Costa Rica. The symptoms are not uniformly distributed in the tree, so some branches are symptomatic while others are not. These symptoms are similar to several leaf scorch diseases caused by the bacterium Xylella fastidiosa Wells (2,4). This bacterium has been detected in coffee and citrus plants in Costa Rica. Of 227 avocado trees tested by double-antibody sandwich (DAS)-ELISA with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) from 2000–2004, 188 were positive. Results of ELISA tests of individual trees varied with the season and branches tested. Fifteen greenhouse-grown, ELISA-negative avocado seedlings were grafted with budwood from an ELISA-positive tree. Eight of these developed scorch symptoms and one also showed chlorotic mottling and deformation, showing that the disease is graft transmitted. All of these features are characteristic of diseases caused by X. fastidiosa (2,4). Transmission electron microscopy of leaf petioles from three field trees positive by ELISA, revealed rod-shaped bacilli approximately 1.6 to 2.0 μm long and 0.3 μm in diameter with a rippled cell wall inside xylem vessels and embedded in a matrix; morphology and measurements that are consistent with those reported for X. fastidiosa (2). DNA extraction and PCR attempts have been limited by mucilaginous sap from avocado. Positive PCR results (approximately 472-bp band) were obtained from two of the grafted seedlings and seven field trees from two distinct geographical locations (Alajuela and San José provinces) with DNA extractions from the plant sap using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) following a modified protocol (1) and nested PCR (3). Four of the PCR products, including one from the grafted seedlings, were cloned and sequenced in duplicate. GenBank sequences EU021997 to EU022000 present 99 to 100% sequence identity to a Pierce's disease strain from California (Temecula1) and 94 to 95% to a citrus variegated chlorosis strain from Brazil (Found-5). Several attempts have been made to isolate the bacterium in ‘periwinkle wilt’ and buffered cysteine-yeast extract media with negative results, probably because of the rapid production of mucilaginous sap when the avocado tissues were sampled. To our knowledge, this is the first report of X. fastidiosa in avocado trees. References: (1) M. J. Green et al. Plant Dis. 83:482, 1999. (2) S. S. Hearon et al. Can. J. Bot. 58:1986, 1980. (3) M. R. Pooler and J. S. Hartung. Curr. Microbiol. 31:377, 1995. (4) A. H. Purcell et al. Phytopathology 89:53, 1999.

Comparative Expression Profiling of Nicotiana benthamiana Leaves Systemically Infected with Three Fruit Tree Viruses

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.

(367) Symptom Expression of Mixed Virus Infections in `Chickasaw' Blackberry

Latent infection of Blackberry yellow-vein associated virus (BYVaV) in `Chickasaw' blackberry has been reported. However, plants with characteristic leaf symptoms, such as vein yellowing, chlorotic mottling, and oak-leaf patterns, have tested positive for BYVaV using reverse-transcription polymerase chain reaction. Experiments were initiated to determine if the symptoms expressed in BYVaV infected `Chickasaw' were caused by mixed virus infections. BYVaV, a recently identified crinivirus, was evaluated for synergistic interactions with Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Raspberry bushy dwarf virus (RBDV). `Chickasaw' blackberry plants infected with BYVaV (single infection) were used as receptor plants to establish mixed virus infections with TRSV and ToRSV transmitted by nematodes and RBDV transmitted by bottle grafts. Characteristic symptoms of multi-virus infection will be presented and discussed.