Steve Haber

Overview of some recent research developments in fusarium head blight of wheat

Abstract This article reviews the recent progress of research on fusarium head blight (FHB) of wheat. It addresses the broad areas of strategies for disease management, biological control, the pathogen (Fusarium graminearum (teleomorph Gibberella zeae)), mycotoxins, the effects of dwarfing genes on FHB severity, quantitative trait loci (QTLs) and new perspectives. Where there are recent reviews on this subject, we have deliberately examined the subsequent literature to provide an update on research. With few resistant cultivars available even now, the main tools to manage the disease remain rotation, varietal selection, disease forecasting and fungicides. A few biocontrol organisms are being considered for commercial application. The pathogens sexual life cycle has been investigated in depth, and with its complete genome sequence known, the pathways and genes controlling the sexual development and ascospore release of F. graminearum are being explored. The 3-acetyldeoxynivalenol chemotype of F. graminearum has increased in prevalence in Canada with attendant risks of higher DON levels in cereal grain. Stringent limits on allowable levels of Fusarium mycotoxins in the food/feed chain have been enacted in Europe and the USA, but regulations for Canada are only at the discussion stage with the Canadian Food Inspection Agency. Efforts to develop FHB-resistant lines proceed apace, as these can be selected in most wheat populations despite the adverse effects of dwarfing genes on FHB severity. While more quantitative trait loci (QTLs) for disease resistance continue to be identified and mapped, new resistant cultivars remain disappointingly few. We present some encouraging early results from an alternative approach based on epigenetics.

Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

Temperature-Sensitive Wheat streak mosaic virus Resistance Identified in KS03HW12 Wheat

Wheat streak mosaic virus (WSMV) infection reduces seed yield and quality in wheat. These losses can be alleviated significantly by exploiting genetic host plant resistance. A new source of temperature-sensitive resistance to WSMV, KS03HW12, and its parental lines (KS97HW29/ KS97HW131//KS96HW100-5) were evaluated in both greenhouse and field conditions. Parental wheat lines were exposed to WSMV pressure under different temperatures in growth chambers to determine the stability of the resistance, and 2 years of field yield trials were conducted to confirm effectiveness. To determine the effectiveness of its resistance against a spectrum of isolates, KS03HW12 was tested against six different WSMV isolates of different geographic origins. Among the three pedigree parents, only one, KS97HW29, was resistant. The parental lines of KS97HW29 are not available for testing; therefore, the presumed origin of the resistance could not be further confirmed. None of the six tested WSMV isolates systemically infected KS03HW12 at 18°C. Yield of KS03HW12 in field tests was not different from healthy controls. Thus, the elite winter wheat KS03HW12 appears to be a stable and effective source of temperature-sensitive resistance to WSMV and should be useful for wheat breeding programs.

Determination of the Complete Amino Acid Sequence for the Coat Protein of Brome Mosaic Virus by Time-of-Flight Mass Spectrometry EVIDENCE FOR MUTATIONS ASSOCIATED WITH CHANGE OF PROPAGATION HOST

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the “type” isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.

Natural infection of sorghum by foxtail mosaic virus in Kansas

Sorghum (Sorghum bicolor) was infected by a mechanically transmissible, flexuous, rod-shaped virus. Antiserum made against the purified virus reacted specifically in enzyme-linked immunosorbent assay to the virus and to the potexvirus foxtail mosaic virus (FoMV), indicating that the sorghum virus was an isolate of FoMV. Comparison of the sorghum isolate (H93) to FoMV PV 139 showed that H93 differed biologically by causing severe symptoms in sorghum, not readily infecting certain barley lines, and causing only faint symptoms in barley. At the molecular level, the capsid of H93 had a mass of 23.9 kDa and 217 amino acid residues compared with 23.7 kDa and 215 residues previously reported for the nucleic acid sequence of FoMV. The amino acid sequences of the two viruses were greater than 96% identical. They varied by having four substitutions, one deletion, and three insertions between residues 66 and 67. This is the first report of natural infection of sorghum by FoMV, thus extending its host range among cereal crops.

Characterization of a distinct Johnsongrass mosaic virus strain isolated from sorghum in Nigeria

Summary.A virus isolated from sorghum in Nigeria has been partially characterized. It was tested by enzyme-linked immunosorbent assay (ELISA) using antisera to Maize dwarf mosaic virus, Johnsongrass mosaic virus (JGMV), Sugarcane mosaic virus strain-MDB, Sorghum mosaic virus, and Zea mosaic virus. A partial host range, symptom phenotypes for selected sorghum lines, and the mass of the coat protein (CP) subunit was analyzed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid (aa) sequence determined by time-of-flight mass spectrometry (TOFMS). The Nigerian isolate was positive in ELISA to only JGMV antiserum. It infected sorghum and smooth brome but not oat or johnsongrass. It caused necrosis in 12 of 13 tested sorghum lines, while the USA JGMV isolate caused necrosis in only one sorghum line. In SDS-PAGE, the mass of the Nigerian virus CP was 3,000 Da smaller than that of JGMV-MDO. Moreover, TOFMS analyses showed that, while residues 1–7 of the CP aa sequence were identical to those of JGMV (GenBank #A27631), and residues 57–293 were almost identical to residues 67–303 of JGMV, the intermediate region exhibited significant differences, including a 10 aa deletion. These data indicate that the virus should be considered a distinct isolate of JGMV (JGMV-N) and expands the known range of JGMV to Africa.

New Sources of Temperature-Sensitive Resistance to Wheat streak mosaic virus in Wheat

Expressing temperature-sensitive resistance (TSR) protects wheat against yield losses from infection with Wheat streak mosaic virus (WSMV). In examining how 2,429 wheat accessions from the National Small Grains Collection responded to inoculation with the Sid81 isolate of WSMV, 20 candidate TSR sources were discovered. To differentiate their relative effectiveness, accession responses over 21 days to inoculation with GH95, Sid81, and PV57 virus isolates in regimes of 18 and 20°C were observed. At 18°C, all 20 candidate TSR sources were uniformly or nearly uniformly asymptomatic 21 days after inoculation with the PV57 isolate, resistance indistinguishable from resistant checks KS96HW10-3 and RonL. By contrast, the Sid81 isolate induced symptoms in low but significant proportions of plants of two candidates, and the GH95 isolate in high proportions for four candidates and low but significant proportions for two others. In the more stringent 20°C regime, the uniform or near-uniform induction of symptoms in response to inoculation with GH95 failed to differentiate among the 20 candidate TSR sources and two resistant checks, while PV57 and Sid81 identified several candidates that performed similarly to KS96HW10-3 and significantly better than RonL. By identifying new sources of resistance, this study contributes to the control of WSMV.

Characterization of the Agent of “High Plains Disease” MASS SPECTROMETRY DETERMINES THE SEQUENCE OF THE DISEASE-SPECIFIC PROTEIN

The “32-kDa” protein specifically associated with high plains disease was characterized by time-of-flight mass spectrometry, after the agent had been isolated in pure culture by “vascular puncture inoculation,” a novel mechanical means of transmission. Two isolates from different geographic locations each consisted of a mixture of subpopulations that were highly homologous to an amino acid sequence derived from a nucleotide sequence (U60141) deposited in GenBank™ by the Nebraska group as “the probable N-protein of high plains virus.” However, the U60141 sequence was found to be incomplete; de novo sequencing of peptides produced by proteolytic digestions of the 32-kDa band from an SDS-PAGE separation showed that an additional 18 amino acid residues were present at the N terminus. BLAST (basic local alignment search tool) examination of the sequence showed no significant homology with any protein in the databases, indicating that the infectious agent of high plains disease is likely a member of a hitherto unclassified virus group.

Effectiveness of three potential sources of resistance in wheat against Wheat streak mosaic virus under field conditions

Wheat streak mosaic virus is an established major threat to wheat in North America and is newly identified in Australia. Three genetic sources of resistance were examined, Wsm1 (from an alien translocation), Wsm2 (from CO960293-2), and c2652 (selected in Canada). We report their effectiveness in the field when inoculated with an Australian WSMV isolate. Also included were advanced breeding lines with and without Wsm2 and a number of elite Australian cultivars. ELISA testing on individual plants indicated we achieved between 85% and 100% infection with WSMV in susceptible lines following artificial inoculation which reduced their yield by 22 to 44% and height by 19 to 51%. Kernel weight was significantly affected in some of the susceptible lines. All three sources of resistance (Wsm1, Wsm2, c2652) and Wsm2 derivatives protected wheat against infection despite repeated inoculation. Inoculated resistant plots were virtually disease free and suffered neither significant yield loss nor height reduction. National yield trials of the breeding derivatives showed no difference in yields between those with and without Wsm2 under non-WSMV conditions.

Association of a virus with wheat displaying yellow head disease symptoms in the great plains

Wheat with yellow head disease (YHD) (yellow heads and mosaic leaf symptoms) has been observed in Kansas since 1997. A pathogen was transmitted from the infected wheat to maize by vascular puncture inoculation and to Nicotiana benthamiana by rub inoculation. The original infected wheat and infected maize and N. benthamiana test plants all produced a unique 32- to 34-kDa protein when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Time-of-flight mass spectrometry analysis of the unique 32- to 34-kDa protein showed that the amino acid sequence was most closely related to the nucleoprotein of Rice hoja blanca virus, indicating that the virus causing YHD symptoms in wheat is a tenuivirus. Antiserum made to this protein failed to react with extracts made from healthy wheat or wheat infected with Wheat streak mosaic virus or the High Plains virus. The antiserum did react to extracts made from symptomatic wheat, maize, and N. benthamiana, shown by SDS-PAGE to contain the unique protein, and to extracts of wheat with YHD symptoms from Kansas, North Dakota, South Dakota, and Oklahoma. The name Wheat yellow head virus is proposed for this virus.