Healthy Leafhoppers Research Articles

Maize as a New Host for Oat blue dwarf virus.

Oat blue dwarf virus (OBDV) is a marafivirus present throughout the North American Great Plains and is found at low incidence levels in barley, oats, and flax in the Upper Midwest United States. Cropping patterns in this region have changed considerably in recent years, with much greater acreages devoted to maize in areas historically planted to small grains. Considering that OBDV is endemic in small grains in this region and that its aster leafhopper vector (Macrosteles quadrilineatus) is commonly detected in maize (2), we questioned whether maize might be a host for OBDV. Although Westdal (4) reported that maize was not a host for OBDV, it is the primary host for the related Maize rayado fino virus (MRFV) transmitted by Dalbulus maidis. MRFV is the type member of the marafiviruses and ranges from South America to the southern United States. To reevaluate the ability of maize to serve as a host for OBDV, viruliferous aster leafhoppers were allowed to feed on young maize seedlings for a 7-day inoculation access period. Plants were assayed for the presence of OBDV by ELISA 21 days after leafhopper removal. Initially, open-pollinated Sunglow sweet corn was tested in three experiments, with infection rates of 17 to 100%. A test of hybrid Sunglow resulted in 63% infection. These are comparable to transmission levels obtained when oat and barley plants are fed upon by viruliferous leafhoppers. Subsequently, 10 additional varieties of sweet and field corn were tested to determine if they could serve as hosts for OBDV. Sweet corn included Silver Queen, Peaches and Cream, Morning Star, Sugar Dots, Kandy Korn, and Golden Cross Bantam. Field corn included four lines provided by the maize breeding program of M. Carena at North Dakota State University (056640, 056643, 056612, and 056652). At least one plant was infected in all sweet and field corn varieties except 056612. Infected plants were largely asymptomatic, although a very faint stipple-striping was observed on some plants. To determine whether leafhoppers could acquire OBDV from infected maize and transmit the virus to other hosts, healthy leafhoppers were fed upon detached leaves of infected maize for 4 days (approximately 30 days after initial infection) and subsequently transferred three times at weekly intervals to barley and oats. Infection rates of 42 to 55% were observed in target plants, indicating efficient transmission from maize to other cereal hosts. The extent of natural infection of maize by OBDV or economic loss, if any, remains to be determined. This discovery extends the host range of OBDV to include maize and suggests that maize has the potential to serve as a natural reservoir for OBDV. The identification of hosts for known and new marafiviruses, such as those recently detected in citrus (3) and grape (1), will expand our understanding of the ecology and epidemiology of viruses within this group.

Comparative studies on alkaline phosphatase (ALP), alkaline phosphatase amplification (AMP) horseradish peroxidase (HRP), penicillinase (PNC) and avidin biotin penicillinase amplification ELISA in detection of maize streak virus (MSV) in maize plants and Cicadulina mbila (leafhoppers) insect vector

Comparative study of dilution end point on various detecting enzymes in ELISA techniques to determine sensitivity level for detection of Maize Steak Virus (MSV), both in the maize plant and the insect vector Cicadulina mbila (leafhopper), was carried out using Alkaline phosphatase (ALP), Horseradish peroxidase (HRP), Alkaline phosphatase amplification (AMP) and Penicillinase (PNC) and Avidin- Biotin Penicillinase amplification based ELISA. Assays with the PNC-based ELISA were as sensitive as analogous assays performed with ALP and HRP for the virus in infected maize plant. The HRP and the Penicillinase- based ELISA were able to detect virus in three and crushed leafhoppers respectively. Only the Avidin- Biotin Penicillinase amplification technique was able to detect viruses in one single leafhopper. As expected, the ALP and the alkaline phosphatase enzymatic amplification systems were not useful in virus detection in the insect vector due to the high background of the healthy leafhoppers. This could be due to insects containing their own phosphatase stem. The protein A coat Penicillinase technique was not useful due to non specific reaction. Key words: Enzyme Linked Immunosorbent Assay, maize streak virus, Cicadulina mbila, alkaline phosphatase, alkaline phosphatase amplification, horseradish peroxidase, penicillinase based ELISA.

Controle químico da cigarrinha-do-milho e incidência dos enfezamentos causados por molicutes

O objetivo deste trabalho foi verificar a eficiência do tratamento inseticida de sementes de milho no controle de Dalbulus maidis e na redução da incidência de enfezamentos em viveiro telado e em campo. Foram realizados dois experimentos; no experimento 1, em viveiro telado, sementes de milho foram tratadas com imidacloprid e thiamethoxan e, nessas plantas, cigarrinhas sadias, cigarrinhas infectantes com fitoplasma ou com espiroplasma foram confinadas. Avaliaram-se eficiência de controle, incidência de plantas com enfezamentos, altura das plantas e produção de grãos. No experimento 2, em campo, imidacloprid e thiamethoxan foram utilizados em tratamento de sementes e pulverizações aos 10 e 20 dias após a semeadura. A incidência de enfezamentos e a produção de grãos foram avaliadas. Em viveiro telado, os produtos imidacloprid e thiamethoxan proporcionaram controle de adultos de D. maidis acima de 50%, até o trigésimo dia, e reduziram a incidência de doenças e danos no crescimento e produção das plantas infectadas expostas às cigarrinhas infectantes aos dois dias após a emergência. Em campo, não foi constatada redução na incidência de enfezamentos ou ganho em produção, possivelmente devido ao fluxo migratório de cigarrinhas infectantes.

Identification of a non-host plant of Xylella fastidiosa to rear healthy sharpshooter vectors

Rearing leafhopper (Hemiptera: Cicadellidae) vectors free of Xylella fastidiosa is a requirement for studies of various aspects of vector-pathogen interactions. The selection of a plant that allows vector development but not bacterial multiplication is desirable to produce healthy vectors. In this study, two leafhopper hosts, Vernonia condensata ('boldo') and Aloysia virgata ('lixeira') were needle inoculated with citrus and coffee strains of X. fastidiosa to evaluate if these plants support pathogen colonization. The inoculated plants did not present symptoms and the pathogen was not detected by culture and PCR tests, neither soon after inoculation (7-14 days) nor later, at 1, 4, 6 and 12 months after inoculation. To obtain healthy adults of the leafhopper vectors Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, Homalodisca ignorata and Oncometopia facialis, early-instar nymphs were reared on V. condensata. X. fastidiosa was not detected in any of 175 adults obtained. V. condensata and A. virgata are nonpropagative hosts of X. fastidiosa and enable the production of healthy leafhoppers for vector studies.

Evidence for the physical integrity of flavescence dorée phytoplasmas purified by immunoaffinity from infected plants or leafhoppers and the plant pathogenicity of phytoplasmas from leafhoppers

Phytoplasmas were extracted from flavescence dorée‐infected broadbean (Vicia faba) using a vacuole isolation medium, and were immunoaffinity purified from infected leafhoppers. Purified phytoplasmas from both host sources were immunolabelled and observed under the electron microscope. The infectivity of the purified phytoplasmas from leafhoppers was checked by injecting healthy leafhoppers which were then allowed to feed on healthy V. faba seedlings. The appearance of typical symptoms in these plants, and the positive results obtained in ELISA with extracts of some of the injected leafhoppers and with symptomatic V. faba, indicated that the purified phytoplasmas had retained their infectivity and had multiplied in the injected leafhoppers which had become infective. These results support a previous report that phytoplasmas purified by immunoaffinity chromatography are well preserved.

Temperature-Dependent Pathogenicity of the X-Disease Mycoplasma-Like Organism to its Vector, Paraphlepsius irroratus (Homoptera: Cicadellidae

The effect of the X-disease mycoplasma-like organism (MLO) on the longevity of Paraphlepsius irroratus (Say) was investigated at four fluctuating temperatures averaging 15, 20, 25, and 30°C. The X-disease MLO in diseased leafhoppers was assayed with a radiolabeled cDNA probe. At the two lower temperatures, the X-disease MLO is pathogenic to P. irroratus . At 15 and 20°C, healthy leafhoppers lived 62 ± 16 ( |$\bar x$| ± SE) and 52 ± 22 d, respectively, whereas X-diseased leafhoppers lived 35 ± 21 and 23 ± 9 d, respectively. In the 25–30°C temperature range, MLO pathogenicity was masked by the temperature effect on leafhopper survival. At 25 and 30°C, longevity of healthy leafhoppers was 15 ± 9 and 7 ± 3 d, respectively, whereas X-diseased leafhoppers lived 14 ± 4 and 7 ± 4 days, respectively. Below 20°C, the MLO accounted for a 32% reduction in leafhopper longevity compared with <6% above 25°C. The MLO acquisition ranged from 32.4 to 36.9% with no difference ( P ≤ 0.05) between temperature regimes.

Morphology and differential counts of hemocytes of healthy and wound tumor virus-infected Agallia constricta

Six types of hemocytes were found in Agallia constricta leafhoppers: plasmatocytes, spherule cells, granular hemocytes, adipohemocytes, oenocytoids, and prohemocytes. Plasmatocytes, spherule cells, and granular hemocytes accounted for 90–95% of all hemocytes in numphs and adult leafhoppers. As the insect aged from second- and third-instar nymphs to 7- and 8-week-old adults, there was a significant decrease in plasmatocytes in healthy leafhoppers compared to wound tumor virus-infected insects. In contradistinction, there were more granular and spherule hemocytes in healthy leafhoppers than in virus-infected ones as the insects aged. In general, there were more prohemocytes in infected than in healthy leafhoppers. Plasmatocytes from 4- to 8-week-old, infected leafhoppers contained large irregularly shaped, cytoplasmic inclusions. Electron microscopy of these cells showed that the inclusions were either large accumulations of wound tumor virus particles or virus-free electron dense bodies.

Multiplication of aster yellows virus in a nonvector leafhopper

Aster yellows virus (AYV) was recovered at various times from a nonvector, Agallia quadripunctata Prov., after the insects were exposed to infected plants. The presence of virus in exposed nonvector insects was demonstrated by injecting extracts of the insects into healthy leafhoppers of the vector, Macrosteles fascifrons (Stål), and then testing the latter for infectivity on aster (Callistephus chinensis Nees.) plants. The virus from exposed nonvector insects was recovered at theendof a 6-day acquisition period and on day 24 after the beginning of the acquisition at a dilution of 16, but not at dilutions of 112 and 124. On day 12 the virus was recovered at all three dilutions. These results showed that in nonvector insects the virus increased in concentration, and then decreased by day 24. Extracts from the intestines of exposed nonvector insects were infective, but preparations from the hemolymph and salivary glands were not. Periodic assays showed that the virus increased in concentration in the intestine, suggesting that this tissue is capable of supporting AYV synthesis. Vector specificity of AYV, therefore, cannot be explained solely on the basis of multiplication in an insect. No virus could be recovered from nonvector insects that were injected with a massive dose of infective AYV.

Studies on the smear technique for detecting virus antigens in an insect vector by use of fluorescent antibodies

The direct and indirect methods with fluorescein-labeled antibodies were compared for the detection of wound-tumor virus (WTV) antigens in smears of leafhoppers Agallia constricta Van Duzee exposed to the virus. In order to obtain specific staining, commercial fluorescein-labeled sheep serum prepared against rabbit gamma globulin for use in the indirect method was passed through a DEAE cellulose column, a procedure known to remove fractions staining nonspecifically. The material that stained specifically by both methods was in the form of particles with brilliant yellow-green fluorescence. WTV-antiserum before and after conjugation had titers of 1 2560 and 1 320 , respectively, showing that some antibodies were either lost or inactivated during the conjugation procedure. For detection of WTV antigens in smears of exposed leafhoppers by the direct method, conjugated WTV antiserum could be diluted only to 1 2 , whereas by the indirect method unconjugated WTV antiserum was satisfactory at a dilution of 1 80 . Also there was much less non-specific fluorescence in smears of leafhoppers stained by the indirect method than by the direct method. Before discovering the superiority of the indirect method of staining the smears, 379 exposed leafhoppers were examined by the direct method. Of these 204 were positive and 134 negative both by smear test (direct method) and by transmission. Eighteen leafhoppers were positive by smear test but did not transmit WTV, and 23 leafhoppers did transmit but were negative by the smear test. Of 319 healthy leafhoppers smeared along with exposed leafhoppers, all, with one exception, which was scored a doubtful positive, were scored negative by the smear test.