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Maize chlorotic mottle virus in Hawaiian-grown maize: vector relations, host range and associated viruses X. Q. Jiang *t, L. J. Meinke*, R. J. Wright*, D. R. Wilkinson* and J. E. Campbell* *Pioneer Hi-Bred International Inc., Plant Breeding Division, Department of Research Specialists, 7250 NW 62nd Avenue, Johnston, Iowa 50131-0085, USA and t Department of Entomology, 202 Plant Industry Bldg, University of Nebraska-Lincoln, Lincoln, Nebraska 685830816, USA

Abstract

Keywords

Studies were conducted on Kauai, Hawaii to identify potential above-ground arthropod vectors of maize chlorotic mottle virus (MCMV), to survey for plants serving as reservoirs for MCMV and also to survey for associated viruses in maize (Zea mays) that might cause corn lethal necrosis disease. MCMV transmission studies used six arthropod species found in MCMV-infected maize fields (Peregrinus maidis, Sardia pluto, Empoasca solana, Adoretus sinicus, Tetranychus sp. and Frankliniella williamsi). After a 1 3-day acquisition feeding period on MCMV-infected maize plants, five arthropod species gave positive test results for MCMV by enzyme-linked immunosorbent assay (ELISA), but only the thrips (F. williamsi) were able to transmit MCMV to healthy maize plants. This is the first evidence of MCMV transmission by thrips. MCMV was found in all maize genotypes tested and was detected by infectivity tests and ELISA in all parts of the maize plant and in mature seed with 13-30% moisture. Fifteen other species of plants tested by ELISA were negative for MCMV, except for one sample of the grass, Trichachne insularis, and one sample of the dicotyledon, Melia azedarach. However, only sap from maize plant parts testing positive for MCMV by ELISA was capable of infecting healthy maize plants. All maize samples from Kauai were negative (by ELISA) for maize chlorotic dwarf virus, wheat streak mosaic virus and maize dwarf mosaic virus strain A (MDMV-A); one maize sample out of 300 tested positive for MDMV strain B. One T. insularis sample tested positive for MDMV-A.

Maize; Frankliniella williamsi; corn lethal necrosis; maize chlorotic mottle virus; maize dwarf mosaic virus; ELISA; vectors

Introduction Maize chlorotic mottle virus (MCMV) was first identified in Peru (Herbert and Castillo, 1973), and has since been reported in Argentina (Teyssandier, N o m e and Daibo, 1983), Mexico (Gordon et al., 1984), and the states of Kansas (Niblett and Claflin, 1978) and Nebraska (Doupnik, Lane and Wysong, 1982), in the United States. Several serotypes have been identified (Uyemoto, 1978; Uyemoto, Bockelman and Claflin, 1980; Gordon et al., 1984). M C M V causes symptoms in maize (Zea mays L.) ranging from a relatively mild chlorotic mottle to severe stunting. Crop losses of 10-15% in natural field infections and up to 59% in experimental maize plots have been reported (Castillo-Loayza, 1977). When M C M V occurs in combination with either maize dwarf mosaic virus strain A or B (MDMV) or wheat streak mosaic virus (WSMV), a more severe disease known as corn lethal necrosis (CLN) results. Crop losses due to C L N have been reported as high as 91% (Niblett and Claflin, 1978). **Towhom correspondence should be addressed 0261-2194/92/0310248-07 ~ 1992 Butterworth-Heinemann Ltd CROP PROTECTION VoI. 11 June 1992

M C M V can be vectored by six chrysomelid beetle species including the cereal leaf beetle [Oulema melanopa (L.)], two flea beetles [Chaetoenema pulicaria Melsheimer and Systena frontalis (Fab.)], and three corn rootworm species (Diabrotica undecimpunctata howardi Barber, D. barberi Smith & Lawrence, and D. virgifera virgifera LeConte) (Nault et al., 1978; Jensen, 1985). Several aphid species, two leaf hoppers, a planthopper, a whitefly, a scarab beetle, and a noctuid larva have also been tested for transmission, giving negative results (Castillo-Loayza, 1977; Nault et al., 1978).

Kauai background Seed companies have been growing maize on the island of Kauai, Hawaii, USA for > 20 years. In some fields, maize has been grown continuously for the last 20 years. Occasionally in the past, plants with virus symptoms have been noted. Those plants were thought by one of us (D.R.W.) to be infected by M D M V or sugar-cane mosaic virus (SCMV), but neither was positively identified. In

Maize chlorotic mottle virus in Hawaii: X. Q. Jiang etal, Table 1. MCMV detected by ELISA in arthropod species collected from MCMV-infected maize fields, Kauai, Hawaii, April 1990 Sample description Field collected

After feeding on MCMV-infected maize

Species

ELISA reading at A4o5 ,,1

Adoretus sinicus, head A. sinicus, a b d o m e n A. sinicus from Acalypha wilkesiana Leptodictya tabida Tetranychus sp. Frankliniella williamsi Sardia pluto Peregrinus maidis Rhinocloa forticornis Empoasca solana Rhopalosiphum padi

1.04" 0.88"

Blank

0.00

P. maidis S. pluto

0.32 0.55"

E. solana Tetranychus sp.

1.58 ° 1.14"

0.25 1.03" 0.38 0.69" 0.03

0.00 0.00 O.13 O. 17

"Positive readings, as defined in Materials and Methods

January 1990, plants with symptoms that resembled CLN were noted in one field in the Mokihana Valley in southwestern Kauai. Samples from this field were sent to Dr B. Lockhart at the University of Minnesota where they tested positive for MCMV and MDMV by serological tests (B. Lockhart, personal communication). An additional 13 samples were sent to Drs L. Lane and S. Jensen at the University of Nebraska-Lincoln. MCMV was identified by polyacrylamide gel electrophoresis, infectivity tests and serology in 11 samples and putative potyviruses were identified in 11 samples (L. Lane and S. Jensen, personal communication). ELISA testing of additional fields of maize on Kauai showed that MCMV was spreading rapidly to the west from the first field confirmed to have MCMV (X. Q. Jiang, unpublished data). The rapid spread of MCMV suggested that the vector might be an arthropod. Thrips were observed to be unusually abundant at this time in infected fields and it was thought that they might be a vector. A sample of thrips, later identified as Frankliniella williamsi (Hood), collected in March tested positive for MCMV by ELISA (Jiang, Wilkinson and Berry, 1990). This indicated that thrips were acquiring the virus, and that further studies were needed to document MCMV/vector relationships on Kauai. This paper reports the results of experiments conducted in April and May 1990 to obtain additional information on MCMV and its potential vectors in maize on Kauai. These studies concentrated on identifying possible above-ground arthropod vectors of MCMV, surveying for plant species serving as reservoirs for MCMV, and surveying for associated viruses in maize that might be capable of causing CLN. Materials and methods ELISA The biotin-labelled indirect ELISA method (Pratt et al., 1986; Clark, Lister and Bar-Joseph, 1988; Harlow and

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Lane, 1988) was used to detect MCMV in all of the tests. IgG was prepared by use of an immunoattinity column (Sigma Chemical Co., St Louis, Missouri), and labelled with biotin (Harlow and Lane, 1988). Samples were ground in porcelain mortars with 2 × (w/v) ELISA extraction buffer [0.01M sodium potassium phosphate buffer, pH7.4, containing 0.02% sodium azide (w/v), 0.8% sodium chloride (w/v), 0.05% Tween-20 (v/v) and 2% polyvinylpyrrolidone mol. wt 40000 (w/v), (Eastman Kodak Company, Rochester, NY)]. Two hundred microlitres of sample/buffer was transferred to fiat-bottomed microelisa plates (Nunc, Kamstrup DK-4000 Roskilde, Denmark) previously coated with 200 lai MCMV immunoglobulin (2 lag ml- ~) and then incubated overnight at 4°C. Purified MCMV at different concentrations was used as a positive control in all tests. After incubating the plates with samples overnight at 4°C, plates were washed three times with phosphate-buffer saline (PBS) containing 0.05% Tween-20. Next, 200 lal biotin-labelled (1 lag ml- ~) MCMV immunoglobulin was added to each well and plates were incubated at room temperature for 1 h. After washing with PBS three times, 200 lal strepavidin-alkaline-phosphatase (Zymed Lab. Inc., S. San Francisco, California) at a 1 : 1000 dilution was added to each well and incubated for I h at room temperature. Next, 200 lal p-nitrophenyl phosphate (0.5 m g m l - 1) (Sigma Chemical Co., St Louis, Missouri) was added to each well and incubated for 10-15 min at room temperature. The reaction was terminated by adding 60 lal 4 N NaOH to each well. The ELISA reactions were measured spectrophotometrically at 405 nm using a Minireader II (Dynatech Laboratories, Inc., Alexandria, Virginia), and positive readings were confirmed by visual observations. The A 4 0 5 n m values presented (Tables 1-3) were obtained by subtracting the buffer control absorbance values (average of three values) from the sample values. Samples from plants and insects were run on the same plates and thus shared positive and negative controls, although data are reported separately below. In all assays, a sample was considered MCMVpositive if the A4o5 n m value was greater than twice the mean value of a healthy, uninfected control plant and exhibited a visible yellow colour. (The minimum positive A4os,m value was 0.40). In all tests, MCMV at concentrations ~0.5lagml 1 produced positive readings.

Arthropod transmission studies The objective of these studies was to determine if aboveground arthropods found in maize on Kauai were capable of transmitting MCMV to maize. Six species ofarthropods [Peregrinus maidis (Ashmead), Sardia pluto (Kirkaldy) (Homoptera : Delphacidae); Empoasca solana DeLong (Homoptera:Cicadellidae); Adoretus sinicus Burmeister (Coleoptera : Scarabaeidae); Tetranychus sp. (Acari : Tetranychidae); and Frankliniella williamsi Hood (Thysanoptera : Thripidae)] were collected on or near maize from several fields in south-west Kauai in sufficient numbers to be used in these studies. Insects were either aspirated

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Maize chlorotic mottle virus in Hawaii: X. Q. Jiang etaL

Table 2. Symptoms of maize chlorotic mottle (MCM) and ELISA readings after mechanical inoculations of healthy maize plants with plant sap or arthropod homogenate from samples positive by ELISA for maize chlorotic mottle virus (MCMV), April - May 1990, Kauai, Hawaii

ELISA reading at A4o5 ,,1 (20 days)

Presence of MCM symptoms (20 days)

Mean

(range)

Plants with MCM symptoms/total plants (25 days)

Treatment

Inoculation

Control inoculations

Buffer MCMV, 50 p.g ml- 1 MCMV, 500 p.gml- 1

No Yes Yes

0.19 1.92~ 1.96a

(0.12-0.29) (1.90-1.94) (1.93-1.97)

0/14 13/13 14/14

Inoculated with plant sap

Maize leaf sap Trichachne insularis tassel

Yes No

1.91" 0.12

(1.83-1.95) (0.02-0.32)

22/22 0/15

Maize, seed, 13% moisture pot 1 pot 2 pot 3 Maize, seed, 30% moisture

No No Yes Yes

0.56~ 0.27 1.87~ 1.81~

(1.71-1.91)

0/5 0/5 2/5 13/13

Yes No

1.86" 0.25

(1.83-1.93) (0.11-0.39)

13/13 0/7

Yes Yes No

0.05 1.97" 1.82"

0/5 1/5 1/4

No Yes No

1.96~ 1.82~ 1.89~

4/4 5/5 I/4

Inoculated with arthropod Frankliniellawilliamsi homogenate F. williamsi + detergent A. sinicus

pot 1 pot 2 pot 3 Tetranychus sp.

pot 1 pot 2 pot 3

aPositivereadings, as definedin Materials and Methods. Mean ELISAvaluesare based on three replicates(pots), exceptwhereindividual replicatedata are reported. Where means are reported, all replicateswere positiveor negative,consistent with the reported mean Table3. Maize chlorotic mottle (MCM) symptoms and ELISA readings from studies to identify arthropod vectors of maize chlorotic mottle virus (MCMV), April - May 1990, Kauai, Hawaii

ELISA reading at A4o5 nm (20 days) Treatment Field-collected with acquisition feeding

Field-collected without acquisition feeding

Species

Presence of MCM symptoms (20 days)

A. sinicus Tetranychus sp. F. williamsi P. maidis S. pluto E. solana

Mean

(range)

Plants with MCM symptoms/total plants (25 days)

No No Yes No No No

0.05 0.03 1.88" 0.02 0.04 0.02

(0.02-0.10) (0.01-0.05) (1.80-1.96) (0.01-0.02) (0.03-0.05) (0)

0/19 0/24 19/19 0/18 0/23 0/14

Yes No Yes

1.84~ 0.14 1.89a

F. williamsi

pot I pot 2 pot 3

4/4 0/5 4/4

Negative control

Uninfested plants

No

0.08

(0.01-0.33)

0/40

Positive control

MCMV, 501agml- 1

Yes

1.80~

(1.76-1.86)

7/7

~Positivereadings, as defined in Materials and Methods. Mean ELISA valuesare based on 2-8 replicates(pots), exceptwhere individualreplicatedata are reported. Where means are reported, all replicateswere positiveor negative,consistent with the reported mean

directly f r o m plants o r swept f r o m plants w i t h a net a n d t h e n aspirated. S p i d e r m i t e s w e r e collected by r e m o v i n g infested m a i z e leaves; m i t e s were later t r a n s f e r r e d by p l a c i n g infested l e a f tissue at the base o f plants a n d a l l o w i n g mites to c r a w l o n t o the maize. E a c h species was collected a n d t r a n s p o r t e d in a single-species c o l l e c t i o n to a v o i d possible m o v e m e n t o f virus b e t w e e n species. A s p i r a tors w e r e w a s h e d w i t h d e t e r g e n t b e t w e e n e a c h use to i n a c t i v a t e viruses.

CROP PROTECTION Vol. 11 June 1992

A r t h r o p o d s used in the M C M V t r a n s m i s s i o n studies were c a g e d o n k n o w n M C M V - i n f e c t e d p l a n t s (as determ i n e d by E L I S A ) t r a n s p l a n t e d f r o m the field i n t o p o t s (1 p l a n t per pot). Plants w e r e at a p p r o x i m a t e l y the V 6 - V 7 l e a f stage (Ritchie, H a n w a y a n d B e n s o n , 1986) ( 3 1 - 6 2 c m tall). C a g e s w e r e m a d e w i t h fine m e s h fabric ( N i t e x n y l o n , p r o d u c t N o . 3-112/50, m e s h o p e n i n g o f 112 I~m, 5 0 % o p e n area, T e t k o Inc., R o l l i n g M e a d o w s , I L 60008) s p r e a d o v e r a wire m e s h frame. F a b r i c was t a p e d to the b a s e o f the p o t s

Maize chlorotic mottle virus in Hawaii: X. Q. Jiang etaL

and pots were held outdoors at the Pioneer Hi-Bred International, Inc. facility (PHIF) near Kekaha, Kauai. Plants were grown in field soil and watered from the bottom. Arthropods were allowed to feed for 1-3 days to acquire virus before being transferred to healthy seedlings. The acquisition feeding periods were as follows: 1 day, P. maidis, S. pluto, E. solana; 2 days, F. williamsi, Tetranychus sp.; 3 days, A. sinicus. After the acquisition feeding period, varying numbers of arthropods (depending on their abundance and estimated damage potential to maize) were transferred to MCMVuninfected (as determined by ELISA) plants in caged pots held outdoors. The following numbers of arthropods were introduced per pot: P. maidis: 21-25; S. pluto: 25-27; E. solana: 14-33; A. sinicus: 10; Tetranychus sp.: 50; F. williamsi: 75. Additional F. williamsi were collected from MCMV-infected maize in the field and transferred to pots ( ~ 50-75 per pot) without an acquisition feeding period, to determine the ability of thrips to vector MCMV when collected directly from infected plants in the field. Arthropods transferred to healthy plants after the acquisition feeding period were all adults with the following exceptions: Tetranychus sp. were ~50% adults and ~50% immatures and F. williamsi were ~90-95% adults. Each pot contained 3-5 maize seedlings of inbred B73, growing in a peat-vermiculite soil mix, and was surrounded by a fabric cage (made with a fine mesh fabric as described above) to exclude insects. Pots were placed in a trough containing at least 2.5 cm of water. The base of the fabric sleeve extended below the base of the pot, and was thus sealed with water. Plants were at approximately the V3 leaf stage (Ritchie et al., 1986) when arthropods were added. Replicated uninfested plants and plants mechanically inoculated with purified MCMV (50 I~gml- 1) were established as negative and positive controls, respectively. A minimum of three replicates (pots) per arthropod species or control was used. Pots were fumigated 4 days after the introduction of arthropods. Fumigation was conducted in an airtight chamber using an aerosol insecticide bomb, DeCon FourGone (Sterling Drug, Inc., Montvale, N J), containing pyrethrins, piperonyl butoxide and DDVP. Preliminary experiments indicated that this treatment did not produce any visible damage to maize plants. Observations indicated that this treatment was highly effective in killing all arthropods except thrips and spider mites. Plants were rated for visual symptoms (presence or absence within pot) of MCMV infection 20 days after initiation of transmission tests. At this time, samples were cut from the leaf tip of one leaf of each plant in a pot and were bulked for ELISA testing. Individual plants in each pot were rated for visual symptoms of MCMV infection again at 25 days. All ratings for visual symptoms of MCMV infection were made by the senior author. To provide supporting data for the transmission experiment, field-collected samples of the six arthropod species tested were also tested by ELISA for MCMV, but without an acquisition feeding period on known MCMV-infected plants. Samples of arthropods (> 10 adult P. maidis, > 10 adult S. pluto, > 10 adult E. solana, individual adult A.

251

sinicus, 50 adult Tetranychus sp., 100 adult F. williams0 were ground in a mortar and pestle and the resulting homogenate was tested for MCMV by ELISA as described previously. A. sinicus (one adult) collected from nonMCMV-infected Acalypha wilkesiana Mull. Arg. was also tested for MCMV by ELISA. P. maidis (47 adults), S. pluto (32 adults), E. solana (37 adults) and Tetranychus sp. (100 mites) were also tested by ELISA for MCMV after the acquisition feeding period was completed. To determine whether the MCMV detected was viable and capable of causing disease, samples of F. williamsi, A. sinicus and Tetranychus sp. (species that tested positive for MCMV by ELISA) were ground in a mortar and pestle using numbers of arthropods similar to those above. The insect homogenate was then mechanically inoculated onto uninfested plants. Controls consisted of inoculations of buffer solution and purified MCMV at 50 and 500 p.g m1-1. All treatments were replicated three times. Cage construction, maize genotype, rearing conditions, and evaluation of MCMV infection were as described above for the arthropod transmission study. Several other insects were also collected from maize, but in insufficient numbers to conduct disease transmission studies. They were therefore ground and the resulting homogenate was used in ELISA tests to determine whether they tested positive for MCMV. The insects tested were bird cherry-oat aphids (80 adults and nymphs) [Rhopalosiphum padi (L.); Homoptera:Aphididae], western plant bugs (five adults) (Rhinocloa forticornis Reuter; Hemiptera : Miridae), and a lace bug (three nymphs) [Leptodictya tabida (Herrich-Schaeffer); Hemiptera : Tingidae]. Samples of all of the above arthropods were preserved in alcohol and identified by B. Kumashiro and P. Conant, Hawaiian Department of Agriculture, Honolulu, Hawaii. Voucher specimens of these arthropods have been deposited in their museum. MCMV survey

Different genotypes of maize as well as maize from several locations in south-western Kauai and one location in north-eastern Kauai were surveyed for MCMV. Plant samples were selected on the basis of apparent symptoms of MCMV infection. All parts of the maize plant (leaves, stem, root, cob, silk, anther, leaf sheath, and seeds of different moisture content) were tested by ELISA for the presence of MCMV. ELISA was run separately on tissues from individual plants, and multiple plants were tested at each location. To document that the MCMV detected was viable and able to cause disease, plant sap from samples testing positive for MCMV by ELISA was mixed with carborundum dust and rubbed on leaves of uninfected (as determined by ELISA) plants to inoculate them mechanically. Inoculations with buffer and purified MCMV (50 and 500 ~tgml- 1) were used as negative and positive controls, respectively. Treatments were replicated three times. Cage construction, maize genotype, rearing conditions, and evaluation of MCMV infection were as described for the arthropod transmission studies.

CROP PROTECTION Vol. 11 June 1992

252 Maize chlorotic mottle virus in Hawaii: X. Q. Jiang et al.

Grasses and weeds found in and around infected maize fields in south-western Kauai were also tested for MCMV. Plant taxonomists at the Hawaiian Department of Agriculture identified the species collected. In all, individual plant samples from 15 species of monocotyledons and dicotyledons were tested by ELISA for MCMV: these were Cynodon dactylon (L.) Pers., Trichachne insularis (L.) Nees, Cyperus sp., Setaria sp., Panicum sp., Pennisetum purpureum Schumach., A. wilkesiana, Mimosa pudica L., Waltheria americana L., Melia azedarach L., Abutilon theophrasti Medic., Ipomoea obscura (L.) Ker-Gawl., Rhynchelytrum repens (Willd.) C. Hubb., Cenchrus ciliaris L., and Commelina diffusa Burman f. Survey of MDMV-A, MDMV-B, WSMV and MCDV

(maize chlorotic dwarf virus) As CLN is caused by a virus complex, other viruses associated with maize in Kauai were also examined. The same samples that were tested for MCMV as described above were also tested for MCDV, MDMV-A and MDMV-B. MCDV and MDMV-A were assayed using double-sandwich direct ELISA (Harlow and Lane, 1988); MDMV-B and WSMV were assayed using biotin-labelled indirect ELISA. A sample was considered positive if the A4osnm value was greater than twice the mean of the healthy, uninfected control value and exhibited a visible yellow colour. (The minimum positive Ago 5 n m value was O.4O).

Results Arthropod transmission studies F. williamsi, A. sinicus and L. tabida collected from maize fields with symptoms of MCMV infection gave positive test results for MCMV by ELISA (Table 1). However, A. sinicus tested negative when collected from a MCMV uninfected plant (A. wilkesiana). Homogenate from field-collected F. williamsi produced typical symptoms of MCMV infection and positive ELISA readings in the mechanically inoculated maize plants (Table2). The addition of a detergent (Tween-20) to the thrips homogenate, which would inactivate MCMV, prevented infection in the maize seedlings. This indicated that MCMV infection caused the symptoms observed, rather than some other component of the homogenate. Homogenate from the field-collected A. sinicus and Tetranychus sp. also caused symptoms of MCMV infection and positive ELISA readings on healthy plants when mechanically inoculated (Table 2). However, for these two species there were some discrepancies between ELISA readings and MCM symptom expression at 20 and 25 days postinoculation. For example, in pot 1 of A. sinicus (Table 2), at 20 days plants were rated positive for symptoms of MCMV infection but were ELISA negative on this date, and at 25 days no infection was evident based on symptoms. This may have been due to a misdiagnosis of symptoms of virus infection at 20 days, as no plants rated positive at 25 days.

CROP PROTECTIONVol. 11 June 1992

The first rating was done on the basis of presence or absence of symptoms of virus infection within the pot, and a single false positive could have produced this discrepancy. In other cases, symptoms of MCMV infection were not apparent at 20 days but did produce positive ELISA values and symptoms were observable at 25 days. All of the healthy maize plants developed symptoms of MCMV infection and were ELISA positive after being caged with F. williamsi that had previously fed on MCMVinfected maize (Table3). A. sinicus, E. solaria, S. pluto, and Tetranychus sp., after feeding on MCMV-infected plants, did not transfer the virus to healthy maize seedlings (Table3), although ELISA results indicated that MCMV was present in the arthropods (Table2). Furthermore, the delphacid, P. maidis, which was ELISA negative after feeding on MCMV-infected plants (Table 1), did not transfer the virus to healthy plants (Table3). F. williamsi collected from an MCMV-infected maize field and caged on plants without an acquisition feeding period, had variable effectiveness in transmitting MCMV to healthy plants (Table 3). MCMV survey

MCMV was detected (by ELISA) at all sites sampled in south-west and north-east Kauai. All maize genotypes tested, including sweet corn [Zea mays var. saccharata (Sturtev.) Bailey], were susceptible to this virus. MCMV was detected in all parts of the maize plant including leaf, stem, root, cob, husk, silk, kernel, seed, anther and sheath. MCMV could be detected in mature maize seed with 13-30% moisture. The 15 other species of monocotyledonous and dicotyledonous plants tested were all negative for MCMV by ELISA except for one sample of T. insularis and one sample ofM. azedarach. However, the possibility cannot be excluded that these positive results were due to contamination with F. williamsi in the sample, as thrips were later observed feeding in T. insularis, or due to dicotyledonous plant background in the case of M. azedarach. Sap from maize plant samples giving positive test results for MCMV by ELISA was capable of producing symptoms of MCMV infection and positive ELISA readings when mechanically inoculated to healthy maize plants (Table2). Plants inoculated with purified MCMV developed similar symptoms and tested positive for MCMV by ELISA. Plant sap from T. insularis, which tested positive by ELISA, did not produce symptoms of MCMV infection in healthy maize seedlings when mechanically inoculated into the seedlings. These seedlings also tested negative for MCMV by ELISA (Table2). Although sap from maize seeds containing 30% moisture caused visual symptoms of MCMV infection and positive ELISA readings in seedlings mechanically inoculated with sap from those seeds, seeds containing 13% moisture showed varying results (Table 2). Survey of MDMV-A, MDMV-B and MCDV

Among the 300 maize samples from Kauai tested, all were negative for MDMV, except for one plant with MDMV-B.

Maize chlorotic mottle virus in Hawaii: X. Q. Jiang et aL

All maize samples tested negative for MCDV. Sixteen random samples from maize were tested for WSMV and all tested negative. Of 15 species of monocotyledons and dicotyledons sampled, only one sample of T. insularis and one sample of A. wilkesiana were infected with MDMV-A.

Discussion This is the first report of a thrips as a vector of MCMV. These results are notable because they document a new order of insects capable of vectoring MCMV. MCMV had previously been reported to be transmitted only by several beetle species belonging to the family Chrysomelidae (Nault et al., 1978; Jensen, 1985). In addition, previous reports of thrips transmission of viruses have been restricted to the tomato spotted wilt disease, caused by viruses of a different group than MCMV (Harris, 1981). F. williamsi was the only arthropod species that could be confirmed experimentally as a vector of MCMV during the research conducted in Kauai. The other reported MCMV vectors were not observed during this study. The outbreak of MCMV on Kauai may represent an unusual event. The weather in Kauai in the winter of 1989-1990 was abnormally wet and cool, with 4.65 inches of rain in December and 14.20 inches of rain in January in south-western Kauai. There was a period of 3 weeks in January when it rained every day and temperatures were cooler than normal. Because of the wet soil in the Mokihana and Waimea Valleys, it was not possible to follow the normal insecticide application schedule to control various insect pests. Both the cool, wet weather and the interruption of scheduled insecticide applications may have allowed F. williamsi populations to increase. Cool, wet weather is reported to favour the growth of F. wiHiamsi (Sakimura and Krauss, 1945). During the same period as that in which the thrips population increased, symptoms of MCMV infection spread from field to field. F. williamsi collected from infected fields in March were tested by ELISA and the results were positive (Jiang et al., 1990). The environment and soil types of Kauai differ from those of Kansas and Nebraska, which previously were the only places in which MCMV had been detected in the USA (Gordon et al., 1984). Although, in Kansas and Nebraska, MCMV behaviour in the soil is currently hypothesized to be important to its persistence and transmission, soil factors did not appear to have an obvious role in MCMV transmission and spread in Kauai. In preliminary studies in Kauai, potted maize plants were placed in an MCMVinfected maize field. A replicated factorial experiment compared soil types (natural field soil and sterilized potting mix) and exposure to arthropods (caged and uncaged pots). After 21 days in the field, the caged maize plants, regardless of soil type, tested negative for MCMV by ELISA. All of the uncaged plants, regardless of soil type, were MCMV positive when tested by ELISA (D. Wilkinson and J. Ooka, unpublished data). This suggested that airborne vectors were more important than soilborne vectors in short-term transmission of MCMV on Kauai. Lack of evidence confirming the existence of other

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vectors should not be interpreted as evidence of the absence of other vectors. Homogenate from the following arthropods tested positive for MCMV by ELISA: A. sinicus, L. tabida, E. solana, S. pluto and Tetranychus sp. In light of the evidence from this study of the wider host range of MCMV vectors, these species should be studied further as potential MCMV vectors. Different species of arthropod vectors of viruses have different optimal acquisition feeding periods. It is possible that use of different acquisition feeding periods would have given additional positive results in the arthropod transmission studies. Additionally, one of the insects studied, P. maidis, is known to be a vector of maize mosaic virus (Shurtleff, 1980) and of maize stripe virus (Kulkarni, 1973). Assuming that F. williamsi was the major vector responsible for the MCMV disease outbreak in 1990, many unanswered questions remain concerning virus-vector relationships in this system. Additional studies are needed to answer questions such as which insect life stages are capable of acquiring and transmitting the virus and how long the virus persists in the insect after acquisition. Additional ecological information is needed concerning potential alternate hosts of both thrips and MCMV and the seasonal pattern of occurrence of vectors and MCMV in different hosts on Kauai. It is interesting to note that maize samples from Kauai in January 1990 tested positive for MDMV (B. Lockhart, personal communication), but the samples in late April indicated a very low level of infection (one sample out of 300 infected with MDMV-B). The seasonality of arthropod vectors of MDMV is not known on Kauai but could influence the severity of economic loss by influencing the occurrence of CLN. F. williamsi has not been well studied and some information from the literature is inconsistent. It is reported to have a broad geographical range, including North and South America, the Hawaiian and the Philippine islands (Moulton, 1948). More specific reported locations include Virginia, and Washington, DC, USA, Cuba and Mexico (Watson, 1923), and New Jersey (Comegys and Schmitt, 1965). It is not known to occur in Iowa (Moulton and Andre, 1935) or Illinois (Stannard, 1968). According to Zimmerman (1948) 'it has been intercepted at Honolulu on green corn from California'; however, it is not known to occur in California (Bailey, 1957). F. williamsiis reported to be 'a grass thrips having a narrow host range' (Sakimura and Krauss, 1945). Moulton (1948) states that it has been reported from corn and sugar-cane; Zimmerman (1948) lists Panicum purpurascens and Sorghum vulgare as additional hosts; however, Comegys and Schmitt (1965) report its hosts in New Jersey to include only broad-leaved plants. Assuming that F. williamsi is the major vector of MCMV on Kauai, several approaches to control MCMV on Kauai should be considered including a maize-free period, insecticidal control of vectors and roguing of diseased plants. However, until more is known about the ecology of F. williamsi and the vector-virus relationship, such as virus persistence in thrips, transmission efficiency, and minimum time required for virus acquisition and transmission by thrips, the success of any approach would be difficult to predict. F. williamsi may be a potential threat to corn

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Maize chlorotic mottle virus in Hawaii: X. Q. Jiang et al.

production in other parts of its distribution, particularly where corn is grown during the winter months, producing the cool, wet weather which is reported to favour its growth and survival.

Harris, K. F. (1981) Arthropod and nematode vectors of plant viruses. A. Rev. Phytopathol. 19, 391-426

Herbert, T. T. and CImtillo, J. (1973) A new virus of maize in Peru. Proc. 2nd Int. Congr. Plant Pathol. Abstr. No. 0072, American Phytopathol. Soc., St Paul, MN

Jensen, S. G. (1985) Laboratory transmission of maize chlorotic mottle virus by three species of corn rootworms. Plant Dis. 69, 864 868

Acknowledgements The authors appreciate the help from staff members of Pioneer's Kekaha station: J. Klepper, D. Lindahl, R. Oyama, E. Elsing and S. Machado. They are grateful to J. Ooka, University of Hawaii and M. Isherwood, Hawaiian Department of Agriculture for help in sample collection, and plant and arthropod identification, respectively, and L. Lane, University of Nebraska, for the polyclonal antiserum against the MCMV-Nebraska strain. They are also grateful to J. Berry, S. Jensen and F. Baxendale for reviewing an earlier version of this manuscript. This work was supported in part by grants from Northrup King, DeKalb Plant Genetics, and Pioneer Hi-Bred International, Inc. This article is Journal Series No. 9600, Nebraska Agricultural Research Division and Contribution No. 757 of the Department of Entomology, University of Nebraska- Lincoln.

Jiang, X. Q., Wilkinson, D. R. and Berry, J. A. (1990) An outbreak of maize chlorotic mottle virus in Hawaii and possible association with thrips. (Abstr.) Phytopathology 80, 1060

Kulkarni, H. Y. (1973) Comparison and characterization of maize stripe and maize line viruses. Ann. Appl. Biol. 75, 205-216 Moulton, D. (1948) The genus Frankliniella Karny, with keys for the determination of species (Thysanoptera). Revta Entomol. 19, 55-114 Moulton, D. and Andre, F. (1935) Four new Thysanoptera, with a preliminary list of the species occurring in Iowa. Iowa St. Coll. J. Sci. 10, 223-234

Nault, L. R., Styer, W. E., Coffey, M. E., Gordon, D. T., Negi, L. S. and Niblett, C. L. (1978) Transmission of maize chlorotic mottle virus by chrysomelid beetles. Phytopathology 68, 1071-1074 Niblett, C. L. and Claflin, L. E. (1978) Corn lethal necrosis - a new virus disease of corn in Kansas. Plant Dis. Reptr 62, 15 19

Pratt, L. H., McCurdy, D. W., Shimazaki, Y. and Cordonnier, M. M. (1986) lmmunodetection of phytochrome: immunocytochemistry, immunoblotting, and immunoquantitation. Mod. Meth. Plant Anal.. New Set. 4, 50-74

Ritchie, S. W., Hanway, J. J. and Benson, G. O. (1986) How a Corn Plant Develops. Iowa State Univ. Coop. Ext. Serv. Spec. Report No. 48, 21 pp

Sakimura, K. and Krauss, N. L. H. ([945) Collections of thrips from Kauai and Hawaii. Proc. Hawaiian Entomol. Soc. 12, 319 331

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