Concentration of Maize Chlorotic Mottle Virus Increased in Mixed Infections with Maize Dwarf Mosaic Virus, Strain B

Coldberg, K.-B.. and Brakke, M. K. 1987. Concentration of maize chlerotic mottie virus increased in mixed infections with maize dwarf mosaic virus, strain B. Phytopathology 77: 162-167. The concentration of rnaize chlorotic mottle virus (MCMV) was up to chlorophyIl and a lower than normal ratio of chloroplast to cytoplasmic 5.4 times higher in plants infected with both MCMV and maize dwarf rRNA. Purified MCMVhadanextinctioncoefficient of6.7cm2mg' at260 mosaic virus. strain R (MDMV-BE. rhan in plants infected with M C M Y nm, an absorption maximum at 258 nm, minimum at 240 nm. and 25% only. The concentration of MDMV-B was the same in doubly and singly RNA. infected plants. Plants infected with both viruses had a reduced level of Additional kqy words: corn lethal necrosis, %ea may.r. In the United States. a combination of maize chlorotic mottle virus (MCMY) and wheat streak mosaic virus (WSMV) or maize dwarf mosaic virus (MDMV), strain A or B, causes a disease known as corn lethal necrosis (CLN) (30). In fields with CLN, yield losses of up to 90% (24) have been reported in north central Kansas and south central Nebraska, where the disease is currently confined despite the fact that all three viruses have different aerial vectors (h,23.26). MCMV is a spherical ssRMA (1 -47 M Da) virus with a capsid protein of 24.6 kDa (22). I t is readily transmitted mechanically and, experimentally, by six species of chrysomelid beetles (18,23). MDMV-B (26). an aphid-transmitted poryvirus. has a capsid protein of 35 kDa. an inclusion body protein of 66 kDa (1 9), and a ssRNA of 3 M Da (17). WSMV is a mite-transmitted, 700-nm flexuous rod with a capsid protein of 45 kDa, an inclusion body protein of 66 kDa (9). and a ssR NA of 2.8 MDa (6). Plants with CCN show severe yellowing of leaves, which develops into necrosis beginning at the tips and edges of the leaves and progressing inward. Plants of many corn lines, particularly in breds. develop a severe, often fatat disease if they become doubly infected when young. 'The primary goal of this study was to determine if the concentrations of M C M V and MDMV-B were higher in plants with mixed infections than in those with singte infections. The effect of The pubhcmtlon costs of this srtlele were defrayed In part by page charge payment. This artlcle must therefore be hereby marked "adv%rt~sement" in accordance w ~ t h 18 U S.C. % 1734 solely to nnd~cate ~ h l s (act This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society387. 162 PHYTOPATHOLOGY the disease on chloroplast and cytoplasmic rRNAs. a biochemical index of cell damage, is also reported because this information was available as a byproduct of the MCMV assay. A preliminary report of these resutts has appeared (14). MATERIALS AND METHODS Viruses. To eliminate possible contaminants, the MCMV stock isolate was obtained from pIants inoculated with a solution removed from the virus zone of a sucrose gradient and diluted to near the dilution end point. M DMV-B (type strain) was obtained from S. Jensen of this department. The viruses were propagated on maize inbred B73Ht plants grown in a greenhouse. B73Ht is moderately susceptible to CLN and has ranked as more susceptible than 60% of inbreds tested in the NCR2 regional uniform inbred nursery over the last 5 yr (Rrakke, unpublished dare). To reduce the chances of contamination, virus cultures were started anew at monthly intervals from dried leaves kept over CaCll at 4 C. Five plants (B73Ht) were grown per 10-cm pot, inoculated by leaf rubbing 1012 days after planting when they were in the early three-leaf stage, and harvested 6. 10, and t 5 days after inoculatian. Sap from virus-infected leaves was used for inoculum after dilution in water to about 1:s for MDMV-B and 1:15 for M C M V and addition of a few percent Celite. MCMV purification. Infected Ieaves were harvested 123 5 days after inoculation and ground in 0.1 M K>HPO4 ( 1 g per 3 ml) in a blender for 2 min. The extract was filtered through cheesecloth and centrifuged in a Beckman J21 R centrifuge with a JA20 rotor for 5 min at 8,000 rpm at 5 C. One-motar CaClz (1/5Q volume) was added to the supernatant, which was stirred for 20 min. and then Published in PHYTOPATHOLOGY 77:2 (1987), pp. 162-167. Published in PHYTOPATHOLOGY 77:2 (1987), pp. 162-167. tions. The MCMV concentration was, therefore, determined by measuring viral R N A present in density gradients of whofe cell RN A preparations. MCMV R N A sedimented faster t han the cytoplasmic and chloroplast rRNAs and was easily detected in whole cell nucleic acid preparations (Fig. 3). The concentration of MCMV genomic R N A resulting from single and mixed infections differed dramatically (Table 1, Fig. 4). The MCMV-infected third and terminal leaves had concentrations much lower than the corresponding CLN-infected leaves. Except For the third leaf 6 days after inoculation, the CLN-infected leaves had 1.7-5.4 times as much MCMY-RNA as did singly infected leaves. All M C M Vand CLN-infected leaves, except the MCMYinfected third leaf. had less MCMV RNA 15 daysafter inoculation than a t 10 days. There was no consistent difference between singly and doubly infected plants in the concentration of MDMY-B determined by Fig. 1. Absorbance (254 nm) scans of centrifuged gradfcnt columns with extracts orcorn leaves o r of purified maizc chlorotic mottle virus (MCMV) . The peaks are at depths expected ror rrec ribosomal o r viral RNA. Peak I rcpresentsrhe 16Sand I8 S rRNAs, pcak 2 thc23 S rRNA,peak 3 the28 S rRNA,and peak4 the MFMV RNA. A. Anextractofadouhly (corn lcthal necrosis) rnfected prant in buffer C (0. I M IN H J ) ~ COI. 0.3 M NHoCI. I mM EDTA, 1 % SDSpH 9.4). %. Anextract ofdoubIy infected plant in NaGPS buffer (0.3 M NaCI, 0.05 NalH Pod. 0. I M glycine. 1 m M EDTA. pH 9.4). C. An extract or healthy corn leaves with purified M C M V added during grinding in buffer C. D, An cxtract of healthy corn leaves with purihed M C M V addcd during grinding In NaGPS. E. Purlfled M C M V diluted in buffer C. F. Purificd M C M V diluted in NaGPS. The same amount of purified virus was present in ssmples placed on gradients C. D, E, and F. representing purified virus from five times the amount of leaf tissue placed an gradtents A and B (demonstrating the loar of virions during purification). The arca of the M C M V R N A pcaks corresponds t o 20, 19, 20, and 19 pgvirus Tor patterns C, D, E,and F, respectively. Centrifuged for 3 hr a1 54.000 rpm at 15 C in a 7.5-30s (w lv ) sucrosc gradicnt in a SW6O Fkckrnan rotor. ELlSA (Fig. 4). The concentrations of MDMY-B were 1.5-2.5 times higher 10 days after inoculation than 6 days after inoculation and fell slightly by 15 days after inoculation. Extracts from MDMV-B-infected plants were partly purified and assayed by SDS-PAGE for inclusion body protein (66 kDa) (19). There was no apparent change in concentration of the 66 kDa protein in singly versus doubly infected plants (data not shown). Effect of virus infection on rRNA and chlorophyll. There was little change in amounts of 23 Sand 28 S rRNAs or In the 23 S/ 28 S r R N A ratio of the M C M V and MDMV-R plants compared with healthy plants (Table 1). However, the amount of 23 S rRNA and the 23 S t 2 8 S rRNA ratio was usually reduced slightly in CLN-