Transmission of Pantoea ananatis and P. agglomerans, causal agents of center rot of onion (Allium cepa), by onion thrips (Thrips tabaci) through feces.

Frankliniella fusca, the tobacco thrips, has been shown to acquire and transmit Pantoea ananatis, one of the causal agents of the center rot of onion. Although Thrips tabaci, the onion thrips, is a common pest of onions, its role as a vector of P. ananatis has been unknown. The bacterium, P. agglomerans, is also associated with the center rot of onion, but its transmission by thrips has not been previously investigated. In this study, we investigated the relationship of T. tabaci with P. ananatis and P. agglomerans. Surface-sterilized T. tabaci were provided with various acquisition access periods (AAP) on onion leaves inoculated with either P. ananatis or P. agglomerans. A positive exponential relationship was observed between thrips AAP duration and P. ananatis (R² = 0.967; P = 0.023) or P. agglomerans acquisition (R² = 0.958; P = 0.017). Transmission experiments conducted with T. tabaci adults indicated that 70% of the seedlings developed center rot symptoms 15 days after inoculation. Immunofluorescence microscopy with antibodies specific to P. ananatis revealed that the bacterium was localized only in the gut of T. tabaci adults. Mechanical inoculation of onion seedlings with fecal rinsates alone produced center rot but not with salivary secretions. Together these results suggested that T. tabaci could efficiently transmit P. ananatis and P. agglomerans.

A soluble form of the Tomato spotted wilt virus (TSWV) glycoprotein G(N) (G(N)-S) inhibits transmission of TSWV by Frankliniella occidentalis.

Tomato spotted wilt virus (TSWV) is an economically important virus that is transmitted in a persistent propagative manner by its thrips vector, Frankliniella occidentalis. Previously, we found that a soluble form of the envelope glycoprotein G(N) (G(N)-S) specifically bound thrips midguts and reduced the amount of detectable virus inside midgut tissues. The aim of this research was to (i) determine if G(N)-S alters TSWV transmission by thrips and, if so, (ii) determine the duration of this effect. In one study, insects were given an acquisition access period (AAP) with G(N)-S mixed with purified virus and individual insects were assayed for transmission. We found that G(N)-S reduced the percent of transmitting adults by eightfold. In a second study, thrips were given an AAP on G(N)-S protein and then placed on TSWV-infected plant material. Individual insects were assayed for transmission over three time intervals of 2 to 3, 4 to 5, and 6 to 7 days post-adult eclosion. We observed a significant reduction in virus transmission that persisted to the same degree throughout the time course. Real-time reverse transcription polymerase chain reaction analysis of virus titer in individual insects revealed that the proportion of thrips infected with virus was reduced threefold when insects were preexposed to the G(N)-S protein as compared to no exposure to protein, and nontransmitters were not infected with virus. These results demonstrate that thrips transmission of a tospovirus can be reduced by exogenous viral glycoprotein.

Tissue Blot Immunoassay for Detection of Tomato spotted wilt virus in Ranunculus asiaticus and Other Ornamentals.

A tissue blot immunoassay (TBIA) was developed to detect Tomato spotted wilt virus (TSWV) in Ranunculus asiaticus tubers and other ornamentals. TBIA was comparable to double-antibody sandwich enzyme-linked immunosorbent assay for accuracy and reliability. A nondestructive sampling method was used with R. asiaticus tubers to determine: (i) the relationship between tuber infection and size; (ii) the distribution of TSWV in tubers; and (iii) the relationship between tuber infection and tuber germination. Small tubers had a higher percentage (44%) of infection than large tubers (19%). When destructive sampling was acceptable, the central stem tissue was the most reliable to test. TSWV infection was associated with a significant reduction of tuber germination. Among the tubers that tested positive for TSWV infection, 48% of those that germinated produced foliage in which TSWV was detected. The remaining 52% of the infected tubers planted that germinated developed into plants that were asymptomatic and in which TSWV was not detectable after germination. Only 4% of tubers that tested negative produced infected plants after germination. Our results indicate that TBIA can be used in TSWV management programs to identify infected plants and to index tuber crops.

Effects of synthetic cecropin analogs on in vitro growth of Acholeplasma laidlawii.

Four synthetic peptides (Peptidyl MIMs; Demeter Biotechnologies, Inc.) were evaluated for their in vitro activity against Acholeplasma laidlawii. Fifty percent effective concentration values ranged from 1 to 15 microM. Three of these compounds are more lethal than cecropin B against A. laidlawii.

Digestive, salivary, and reproductive organs of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type.

A microscopic analysis of the morphology and ultrastructure of the digestive, salivary, and reproductive systems of adult Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type was conducted using light, scanning, and transmission electron microscopy. The internal anatomy of B. tabaci was found to be similar to that reported for Trialeurodes vaporariorum. In a microscopic analysis of the salivary glands, we have shown that each primary salivary gland is composed of at least 13 cells varying in morphology and staining differentially, while the accessory salivary glands are composed of four morphologically similar cells. We analyzed the course of the alimentary canal in B. tabaci, demonstrated the internal morphology of the organs, and clarified the location of the filter chamber relative to other organs in the whitefly. Our observations confirm that the pair of structures extending from the connecting chamber are caeca that may aid in fluid movement through the midgut and are not Malpighian tubules, as previously suggested. We confirm an earlier finding that the whitefly lacks Malpighian tubules, having instead specialized Malpighian-like cells within the filter chamber at the juncture with the internal ileum. Finally, we provide the first scanning electron microscopic analysis showing the reproductive organs of B. tabaci. Our investigation provides clarified terminology for several components of the digestive and excretory system. We also provide drawings and micrographs that will aid future researchers in localizing the internal organs of B. tabaci. We expect our analysis to provide a valuable tool for studying B. tabaci / plant virus interactions and physiological and biological aspects of this insect.

Characterization of cucurbita maxima phloem serpin-1 (CmPS-1). A developmentally regulated elastase inhibitor.

We report on the molecular, biochemical, and functional characterization of Cucurbita maxima phloem serpin-1 (CmPS-1), a novel 42-kDa serine proteinase inhibitor that is developmentally regulated and has anti-elastase properties. CmPS-1 was purified to near homogeneity from C. maxima (pumpkin) phloem exudate and, based on microsequence analysis, the cDNA encoding CmPS-1 was cloned. The association rate constant (k(a)) of phloem-purified and recombinant His(6)-tagged CmPS-1 for elastase was 3.5 +/- 1.6 x 10(5) and 2.7 +/- 0.4 x 10(5) m(-)(1) s(-)(1), respectively. The fraction of complex-forming CmPS-1, X(inh), was estimated at 79%. CmPS-1 displayed no detectable inhibitory properties against chymotrypsin, trypsin, or thrombin. The elastase cleavage sites within the reactive center loop of CmPS-1 were determined to be Val(347)-Gly(348) and Val(350)-Ser(351) with a 3:2 molar ratio. In vivo feeding assays conducted with the piercing-sucking aphid, Myzus persicae, established a close correlation between the developmentally regulated increase in CmPS-1 within the phloem sap and the reduced ability of these insects to survive and reproduce on C. maxima. However, in vitro feeding experiments, using purified phloem CmPS-1, failed to demonstrate a direct effect on aphid survival. Likely roles of this novel phloem serpin in defense against insects/pathogens are discussed.

Immunoprecipitation of a 50-kDa protein: a candidate receptor component for tomato spotted wilt tospovirus (Bunyaviridae) in its main vector, Frankliniella occidentalis.

A 50-kDa protein that binds to viral particles in solid-phase assays and that is recognized by anti-idiotypic antibodies made against anti-viral glycoproteins G1/G2 (anti-Ids) has been proposed as a receptor candidate for tomato spotted wilt tospovirus (TSWV) in its main thrips vector, Frankliniella occidentalis Pergande (Bandla et al., 1998. Phytopathology 88, 98-104). Here we show the immunoprecipitation of the 50-kDa protein by anti-Ids and by an anti-G1/G2-TSWV conjugate - a new immunoprecipitation method. In addition, we show that anti-Ids made against anti-G1 (anti-IdG1) block virus replication in an insect tissue replication assay. The results indicate that (a) the TSWV-50-kDa protein interaction occurs in solution, as it must do in vivo; (b) G1 is a viral attachment protein; and (c) the 50-kDa protein is a candidate host factor essential for TSWV entry. These results provide additional support for the role of the 50-kDa thrips protein as a viral receptor. Additionally these experiments provide the basis for testing saturable binding and represent an important step toward the first cloning and identification of a cellular receptor for a plant virus.

The nematode resistance gene Mi of tomato confers resistance against the potato aphid.

Resistance against the aphid Macrosiphum euphorbiae previously was observed in tomato and attributed to a novel gene, designated Meu-1, tightly linked to the nematode resistance gene, Mi. Recent cloning of Mi allowed us to determine whether Meu-1 and Mi are the same gene. We show that Mi is expressed in leaves, that aphid resistance is isolate-specific, and that susceptible tomato transformed with Mi is resistant to the same aphid isolates as the original resistant lines. We conclude that Mi and Meu-1 are the same gene and that Mi mediates resistance against both aphids and nematodes, organisms belonging to different phyla. Mi is the first example of a plant resistance gene active against two such distantly related organisms. Furthermore, it is the first isolate-specific insect resistance gene to be cloned and belongs to the nucleotide-binding, leucine-rich repeat family of resistance genes.

Transmission of Pineapple Mealybug Wilt-Associated Virus by Two Species of Mealybug (Dysmicoccus spp.).

ABSTRACT Closterovirus-like particles associated with mealybug wilt of pineapple were acquired and transmitted by the pink pineapple mealybug, Dysmicoccus brevipes, and the gray pineapple mealybug, D. neobrevipes. Mealybugs acquired pineapple mealybug wilt-associated virus (PMWaV) from infected pineapple plants or detached leaves. The virus was detected in plants by tissue blot immunoassay and confirmed by immunosorbent electron microscopy. Plants exposed to mealybugs reared on PMWaV-free pineapple tissue remained uninfected. The presence of ants was correlated with an increased rate of virus spread when caged with D. brevipes. All stages of D. neobrevipes acquired PMWaV, although vector efficiency decreased significantly in older adult females. The probability of a single third-instar immature transmitting the virus was 0.04. Both species of mealybug acquired and transmitted PMWaV from infected pineapple material that had been clonally propagated for decades, and both species acquired PMWaV from sources previously infected with the virus by the other mealybug species.

Interaction of Tomato Spotted Wilt Tospovirus (TSWV) Glycoproteins with a Thrips Midgut Protein, a Potential Cellular Receptor for TSWV.

ABSTRACT Interactions between viral and cellular membrane fusion proteins mediate virus penetration of cells for many arthropod-borne viruses. Electron microscope observations and circumstantial evidence indicate insect acquisition of tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) is receptor mediated, and TSWV membrane glycoproteins (GP1 and GP2) serve as virus attachment proteins. The tospoviruses are plant-infecting members of the family Bunyaviridae and are transmitted by several thrips species, including Frankliniella occidentalis. Gel overlay assays and immunolabeling were used to investigate the putative role of TSWV GPs as viral attachment proteins and deter mine whether a corresponding cellular receptor may be present in F. occidentalis. A single band in the 50-kDa region was detected with murine monoclonal antibodies (MAbs) to the TSWV-GPs when isolated TSWV or TSWV-GPs were used to overlay separated thrips proteins. This band was not detected when blots were probed with antibody to the non-structural protein encoded by the small RNA of TSWV or the TSWV nucleocapsid protein, nor were proteins from nonvector insects labeled. Anti-idiotype antibodies prepared to murine MAbs against GP1 or GP2 specifically labeled a single band at 50 kDa in Western blots and the plasmalemma of larval thrips midguts. These results support the putative role of the TSWV GPs as viral attachment proteins and identified potential cellular receptor(s) in thrips.

Use of a Tissue Blotting Immunoassay to Examine the Distribution of Pineapple Closterovirus in Hawaii.

Specific monoclonal antibodies made to a pineapple closterovirus (PCV) were used in a tissue blotting immunoassay (TBIA) for the detection of PCV in pineapple. More than 2,000 samples were tested in 5 days by one person using this rapid and reliable assay. A survey was conducted using this assay to test more than 20,000 Hawaiian pineapple samples for the presence of PCV. PCV was detected in symptomless pineapple plants in the field and in the USDA pineapple germ plasm collection. Studies of the association of PCV with mealybug wilt of pineapple (MWP) suggest that PCV may be involved in MWP.

Rapid fixation and embedding method for immunocytochemical studies of tomato spotted wilt tospovirus (TSWV) in plant and insect tissues.

A new rapid fixation and embedding technique using microwave energy was evaluated for immunolabelling and examination of ultrastructure of plant and insect cells. Tissues in gluteraldehyde-paraformaldehyde were fixed for fifteen seconds in a microwave at 100% power, and dehydrated. Microwave energy was then used to polymerize the London Resin White (LR White) acrylic resin during the embedding process. Embedded specimens were then thin sectioned (90 nm) and treated with anti-tomato spotted wilt tospovirus (TSWV) antiserum followed by protein A-gold label, or antisera against a TSWV encoded nonstructural protein followed by goat anti-rabbit gold label. Using this technique, structural and nonstructural proteins of TSWV were readily detected and specifically labelled in cells of the insect vector, the western flower thrips, Frankliniella occidentalis (Pergande), and in infected cells of the plant species, Emilia sonchifolia L.