Progress and Prospects of Studies on Polymyxa Graminis and Its Transmitted Cereal Viruses in China＊

PROGRESS IN NATURAL SCIENCE V ol .15 , No .6 , June 2005

REVIEW ARTICLE

Progress and prospects of studies on Polymyxa graminis and its transmitted cereal viruses in China＊

CH EN Jianping ＊＊ (Virology and Biotechnology Institute , Zhejiang Academy of Agricultural Sciences, Key Laboratory of Plant Virology , Ministry of Agri- culture and Zhejiang Province, Hangzhou 310021, China)

Received October 8 , 2004 ;revised October 25 , 2004

Abstract Polymyxa gram inis is a eukaryotic obligate biotrophic parasite of plant roots that belongs to a poorly studied discrete taxonomic unit informally called “ plasmodiophorids” .P .graminis is nonpathogenic , but has the ability to acquire and transmit nine plant viruses w hich belong to genera Bymovirus and Furovirus and cause serious diseases in cereal crop speciesand also result in significant yield reductions in China and elsew here.Genus Bymovirus contains barley yellow mosaic virus (BaYMV), barley mild mosaic virus (BaMMV), w heat yellow mosaic virus (WYMV), w heat spindle streak mosaic virus (WSS MV), and oat mosaic virus (OMV), and genus F urovirus contains soil-borne w heat mosaic virus(S BW MV), oat golden stripe virus(OGSV), and new ly identified Chinese w heat mosaic virus(CWM V)and soil-borne cereal mosaic virus(S BCMV).All these viruses have been sequenced and their w orldw ide distribu- tions have been studied .The viruses are protected by the environment w ithin P .gra minis resting spores that may remain dormant but vi- able for decades(probably until a suitable host plant is encountered).S pontaneous deletion mutants of S BW MV , OGS V and OMV are detected , and these deletion mutants are not transmissible by the fungus.The persistent, soil-borne nature of these diseases makes the use of virus-resistant crop varieties cu rren tly the only practical and environmentally friendly means to control them , and a large number of disease resistant germ plasms have been screened .

Keywords: Polymyxa graminis, cereal viruses, genus Bymovirus, genus Furovirus .

As early as in the 1920s , a mosaic or rosette disease gal vector are ex tremely tolerant to a poo r environ- w as first reported on winter wheat in America , and then ment and chemicals , and they can survive in the soil it w as found that the pathogen w as transmitted via soil. fo r decades ;(2)there are v arious species of viruses In 1925 , Mckinney successfully transmitted the pathogen and strains , and the continuous appearance of new from the infected plants to healthy plants by mechanical pathogenic types and strains breaks the resistance of inoculation and proved that the pathogen w as a virus , the virus-resistant cultivars and creates difficulties in named soil-bo rne w heat mosaic virus (SBWMV).In cereals breeding .Therefore , to understand the mech- 1969 , Rao and Brakke found that SBWMV w as anism of interaction among the viruses , fungal vector [ 1] transmitted by Polymy xa graminis in soil .In later and cereal crops at the molecular level , and to estab- years , similar diseases were also repo rted in Japan , I- lish new sy stem s and methods fo r disease control are taly , France , Germany , Brazil and Argentina , w hich significantly important .Since 1985 , w e have studied attracted people' s attention because of their serious the fungal vector P .graminis , virus species , ge- effects on production of cereal crops . nomic organization , disease epidemiology , fungal In China , Poly myxa graminis transmitted cere- transmission, as w ell as screening of resistant al viruses w ere first identified in the 1970s[ 2] .The germ plasms .In this review , I summarize the re- diseases caused by these viruses have seriously oc- search prog ress and prospects of the fung al transmit- curred in estimated 200 million ha of successive barley ted cereal viruses in China . and w heat crops in Henan , Sichuan , Hubei , Shaanx- 1 Polymyxa graminis transmitted cereal i , Shandong , Jiangsu , Zhejiang , Anhui provinces and Shanghai City and resulted in a loss of 1 .5 million viruses tons of yield .The rapid spread of the diseases and se- 1 .1 Taxonomy and genomic organization rious damage of crops are caused by the follow ing reasons :(1)the thick-w alled resting spo res of the fun- All of the Polymy xa graminis transmitted cereal

＊ Supported by the National Natural S cience Foundation of China (G rant No .30225031) ＊＊ E-mail:Jpchen2001 @Hzcnc.com 482 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .6 2005 viruses contain tw o single-stranded positive-sense reading frame (ORF)w hich encodes a 257 —271 kD RNA genomes , and belong to genera By mov irus and single poly peptide .From this single large poly protein Furovirus respectively . precursor , 8 mature functional proteins , P3 , 7K , CI , 14K , NIa-Vpg , NIa-Pro , NIb and CP from N- M embers of genus By mov irus are mainly dis- terminus to C-terminus , are derived by proteolytic tributed in Asia , Europe and No rth America , and are cleavage (Fig .1(a)).Among these pro teins , NIb is barley yellow mosaic virus (BaYM V), barley mild related to genome replication , C I is possibly associat- mosaic virus (BaMM V), w heat yellow mosaic virus ed to virus movement , NIa-Pro encodes main viral (WYM V), w heat spindle streak mosaic virus (WSS- proteinase , N Ia -VPg encodes genome-linked pro- M V)and oat mosaic virus (OMV).These viruses se- tein , and CP is the only structure protein for RNA riously infect winter barley , w heat , oat and rye crops encapsidation .RNA2 also contains a large ORF and and are mostly serologically related , but have sig nifi- encodes a 98 —101 kD polypeptide w hich produces cant differences in nucleotide sequences as w ell as host two functional proteins (P1 and P2)after proteolytic ranges[ 3 —8] .Recently , w e have identified that those cleavage .Of these two pro teins , P1 is a proteinase , viruses that occurred in China are BaYM V and BaM- and P2 is possibly associated to fungal transmission . M V for barley and WYMV for w heat .The genomic Both RNA1 and RNA2 contain a poly(A)tail at their o rganization has been determined fo r BaYMV and 3′-terminus[ 9 , 10] .After infection by these bymovirus- W YMV and partial for BaMM V , showing a similari- es, a large number of membranous bodies connected ty among them and some homology to the insect w ith endaplasmic reticulum and cylindrical o r pin- transmitted poty viruses .BaYM V and WYMV , the w heel inclusion bodies fo rm in the infected cells of the w ell studied bymoviruses , have bipartite genomes cereal crops .This cytopatholog ical change can be w ith a larger RNA 1 (about 7 .5 kb)and a smaller used as a diagnostic character for infection of by- RNA 2 (about 3 .5 kb)in the longer and shorter par- [ 4 , 5 , 11] moviruses . ticles, respectively .RNA1 contains one large open

Fig .1 . Genomic organizations of genera Bymovirus(a)and F urovirus(b).

Genus Furov irus has not been classified to any bers and structures of RNA genome , Torrnace and family of plant virus .This genus used to be consid- M ayo re-classified the previous genus Furov irus into ered to contain all fungus transmitted rod-shaped four new genera including Benyv irus , Pomov irus , viruses.According to species of fungal vector , num- Pecluv irus and Furovirus . The new genus Prog ress in Natural Science Vol.15 No .6 2005 w ww .tandf.co .uk/journals 483

Furovirus is characterized by rod-shaped virus parti- w as w idely adopted because its short g row ing period cles, bipartite RNA genomes containing CP-RT and hig h yield made it particularly suitable for use in a gene , but not TGB genes and poly (A) tail , and cropping system having three crops each year (rice transmitted by P .graminis .The definite members from M ay-August and again August-November , fol- of this genus are SBWMV and sorghum chlo rotic spot low ed by either barley , w heat o r oilseed rape from virus (SrCMV), and oat golden stripe virus (OGSV) November-May).This system became widely estab- w as classified as a strain of SBWM V[ 12] .Recently , lished during the 1960s in the middle and lower re- our studies demonstrate that OGSV is an independent gions of the Yang tze River basin and other parts of member[ 13] , and tw o new identified species, Chinese Eastern China .How ever , Zhaoshu 3 proved to be w heat mosaic virus (CWMV)and soil-bo rne cereal very susceptible to BaYM V , and by the mid-1970s, mosaic virus (SBCMV ), are different from SB- BaYM V had become serious in all the areas w here the WMV , and are also members of this genus[ 14, 15] . cultivar w as regularly grow n[ 17, 18] .

Virus particles of genus Furov irus consist of two We have determined the complete sequence of single-stranded positive-sense RNA genomes (Fig .1 BaYM V Yancheng isolate[ 9] .Sequence com parison a- (b))and a coat protein .Both RNA1 and RNA2 are mong Chinese , Germ an and Japanese BaYMV isolates necessary for virus infection and replication , and con- indicated that the 5′-UTR regions have the most sig- tain a cap structure (M 7GpppG)at their 5′-termini , nificant variation , and P1 , P3 , CI , NIa , 5′-part of but no poly(A)tail at the 3′-termini .RNA1 contains CP and 3′-terminus of RNA1 also present different three ORFs .The first one encodes a 149 —153 kD variations .In general, RNA2 show s a g reater varia- polypeptide and the opal termination codon (UGA) tion than RNA1 .The P2 fragment w as more variable can be partially suppressed to ex tend to the second than the CP and phylogenetic analysis of both regions ORF which produces a 208 —212 kD read-throug h show ed that Asian and European isolates fo rm distinct protein .The motifs for methyl-transferase and N TP- clusters, indicating that molecular evolution of binding helicase activity are identified in the 149 — BaYM V isolates is linked to their geog raphical distri- 153 kD protein whereas that for RNA-dependent bution[ 9] . RNA polymerase (RdRP)in the read-through portion .The third ORF at the 3′-terminal region en- Streatley isolate of BaMM V in the UK w as orig- [ 19] codes a putative protein of 36 —37 kD that is believed inally considered to be a strain of BaYMV .In to be a movement protein .RNA2 also has three pre- China , this virus was first detected in Rudong , H aian [ 6, 18] dicted ORFs potentially encoding pro teins of 19 , 84 and Nantong of Jiangsu Province in 1991 .The and 18 —19 kD .The first ORF encodes the coat pro- virus is alw ay s present in association w ith BaYMV in tein .There is an in-frame CUG codon upstream of Chinese BaYM V susceptible barley cultivar , Yan- the first AUG , w hich is believed to initiate a large fuaizhao 3 and Zhaoshu 3 but not in 16 European and coat protein of 25 kD , adding 40 extra amino acids to Japanese cultivars tested , indicating that the the N-terminus of the norm al coat protein .The coat pathogenicity of Chinese BaMMV differs from protein gene finishes w ith an opal codon that is proba- Japanese and European ones , and a Chinese strain of [ 18] bly read through to generate an 84 kD product (o r BaMM V has long been established .Using the 88 kD if it is initiated at the earlier CUG codon)that GCG Pileup prog ram , the available BaM MV coat incorporates O RF2 .The final ORF encodes a putative protein sequences fell into three distinct groups :(1) 18 —19 kD cy steine-rich protein of unknow n func- Chinese , Korean , Japanese (Nal) isolates;(2 ) tion[ 13, 15 , 16] . Japanese (Kal), Germ an isolates ;and (3)UK and French isolates[ 8] . 1 .1 .1 Barley yellow mosaic virus and barley mild mosaic virus BaYM V w as first reported in Japan 1 .1 .2 Wheat yellow mosaic virus and w heat spindle in 1940 and has become a severe disease of w inter streak mosaic virus Yellow mosaic of w heat was w heat in Japan , China , Korea and northwest Eu- first described by Saw ada in 1927 in Japan , and the rope .In China , BaYM V w as first recorded from agent w as identified as wheat yellow mosaic virus Zhuhai Farm of Ninghai County , Zhejiang Province (WYMV)by Inouye in 1969 .It had been confused in the 1950s .However , the disease only became seri- w ith a similar virus w hich w as first identified in 1960 ous in the mid-1970s as a consequence of the intensive in Canada as wheat spindle streak mosaic virus use of a new barley cultivar , Zhaoshu 3 .This cultivar (WSSM V).These viruses have also been reported in 484 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .6 2005

China , India , America , Italy , France and Germany . proteins , N Ib protein is most conservative , and varia- Because of the similarities of biological and serological tions are mainly distributed in the N-terminus of CP , properties, transmissibility and particle morphology C-terminus of CI , 14 K and P1 proteins .It appears of these viruses , Usugi and Saito suggested that these that most of the P2 region on RNA2 is deleted during viruses were , at best , strains of the same virus and in repeated mechanical transmission of the isolate .The the absence of any serological differentiation , and it 3′-UTR of RNA2 is very long (1262 nt)and proves w as probably best to treat them as sy nony mous[ 20] . to have a 532 nt slightly overlapping repeat (99 .1 % Therefore , both WYMV and WSSMV w ere used to identical nucleotides).This is similar to results w ith describe similar viruses that occurred in w heat and rye BaMM V , in w hich a direct imperfect repeat of 522 nt in China and Germany . in the 3′-U TR w as identified from a sub-population of an isolate with a part of the P2 coding region delet- To study the variation between isolates and to ed[ 22] .The occurrence of both deletion and duplica- resolve the uncertainties about the identity of the tion in the same isolate suggests that the laboratory virus in some countries, we have collected 20 infected isolate is more competitive w ithout P2 region needed w heat samples from China , Europe and North Ameri- fo r fungus transmission but there is some disadvan- ca including a rye isolate from Germ any , w hich w as [ 21] tage if the w hole RNA2 becomes sho rt .Presumably identified as WYMV and a typical isolate of WSS- both features arise from detachment and re-initiation M V during 1997 —1999 .In RT-PCR , using primers by the RNA-dependent RNA-polymerase[ 23] . designed from a partial sequence of a French isolate (tentatively described as WSSM V), the fragments of 1 .1 .4 Soil-bo rne wheat mosaic virus and Chinese genomic cDNA w ere consistently amplified from the w heat mosaic virus SBWM V w as first recognized Italian and French isolates but not from the Chinese in Illinois and Indiana in 1919 and identified as the ones .Conversely , products were consistently ob- cause of a majo r disease of winter w heat in 1923 .In tained from the Chinese isolates but not from the Ital- 1993 , we determined the complete RNA2 sequence of ian or French ones, when using primers designed an Oklahoma field isolate of SBWM V , and then con- from the sequence of a Japanese isolate of WYMV . structed the full leng th cDNA clone of it[ 24] . Nucleotide sequences w ere also obtained from regions at or near the 3′-terminus of RNA1 of the 16 Chinese In China , a virus w ith similar particle morpholo- isolates and 4 European and North American isolates , gy and serologically related to SBWMV has been usually including the coat protein gene .Nucleo tide know n for the last tw enty years from Shandong and amino acid sequence comparisons demonstrated Province where it is alw ay s associated w ith [ 25] that the European and North American isolates were WYM V .We have now determined the complete ex tremely similar and were WSSMV w hile the Chi- nucleotide sequence of an isolate of this virus from nese isolates were close to the Japanese isolate and Yantai, Shandong Province , and analysis show s that w ere thus WYM V[ 7, 21] .The homology betw een it only shares 76 .1 % homology in coat protein amino W YMV and WSSMV g roups w as lower than 70 % acid sequence and 71 .1 % full nucleotide sequence and obviously they are different viruses .In addition , w ith SBWMV , therefore it is different from SB- w e have identified 2 strains (Yaan and Yangzhou)of WMV , and is identified as a new member of the W YMV based on their differences of pathogenicity to genus Furov irus , named Chinese wheat mosaic virus [ 14 , 15] some w heat cultivars , and determined their complete (CWMV) .Later , w e have also determined the nucleo tide sequences .Sequence analy sis show s that nucleotide sequences of an isolate of CWM V from the Yang zhou strain is more similar to the Japanese Rong cheng , Shandong Province , w here the infected isolate[ 10] . w heat crop developed particularly severe sy mptom , but the tw o RNAs shared 95 .5 % identity to those of 1 .1 .3 Oat mosaic virus OMV w as first reported the Yantai CWM V isolate[ 26] . in America in 1946 , and now also occurs in the UK , France , Ireland and possibly New Zealand .We have 1 .1 .5 Oat golden stripe virus and soil-borne cereal determined the complete sequence of a UK isolate of mosaic virus OGSV w as first reported in the UK OMV[ 22] .Sequence comparison show s that OMV in 1977 .Since it is serologically related but differs in proteins are almost equally similar to the homologous host range from SBWM V , it has ever been classified proteins of BaYMV , WSSMV and W YMV but more as a strain of SBWMV[ 27] .OGSV causes oat disease distantly related to BaMM V .Among the 10 viral in France , Britain and the USA .In Europe , there is Prog ress in Natural Science Vol.15 No .6 2005 w ww .tandf.co .uk/journals 485 also a P .gram inis transmitted rod-shaped virus particles or RNA form to invade the host cells .How- from w heat and rye and it had been thoug ht for many ever , the viral RNAs and coat proteins can be detect- years to be SBWM V based on similar features of fun- ed prio r to the symptoms development on the leaves. g al transmission , sym ptom , serology and particle The symptoms on infected leaves of cereals usually morphology . appear from late December to early M arch (Fig .2(a) and 2(b)).The first sy mptoms on new leaves are Recently , w e have determined the full sequences chlo rotic spots , w hich then develop gradually to be- of OGSV and the w heat infecting furovirus from come yellow and mosaic stripes.In addition , necrosis France and Italy .Bo th viruses have a similar genomic symptom also develops in March to April in some bar- o rganization to SBWMV and CWM V , but have less ley cultivars .With temperature rising in M ay , new than 70 % nucleotide identical to them , and RNA2 g rowing leaves are sy mptomless and the sy mptoms on has greater differences than RNA1 .Obviously , these infected old leaves g radually disappear , however , the tw o viruses are different from SBWM V , and are sep- [ 13] infected plants are stunt with few er tillers and sm aller arate new members of genus Furovirus .The g rain heads .Loss of grain yield , usually 10 %— French and Italian isolates have been named European [ 13] 90 %, depends on the deg ree of virus infectio n , as w heat mosaic virus (EWMV) .Phylogenetic anal- well as cultivar , climate condition and type of soil. y ses supported the recog nition of these isolates as dis- [ 13] Temperature controls grow th of plants and movement tinct viruses in the genus Furov irus .Meanw hile , of the viruses from roots up to leaves , therefore is the Dr .Koenig and her co-w orkers have also determined main factor fo r disease development and sy mptom ap- the sequences of three isolates of fungus transmitted pearance .Sy mptom appearance also differs on differ- rod-shaped virus from South Germany , of which two ent cultivars of cereal crops , usually , the susceptible isolates w ere from rye and the o ther one from w heat , cultivars show servere symptoms w hereas the resis- and named them soil-bo rne ry e mosaic virus (SBR- [ 28] tant ones show mild even no symptoms .Successive M V) . Sequence com parisons demonstrate that g rowing of susceptible cultivars on a large scale is the there are substantial similarities betw een EWMV main reason for outbreak of the diseases . from w heat in France and Italy and the SBRMV from rye o r wheat in Germany .These isolates are best re- g arded as the same virus and clearly distinct from SB- WMV , OGSV and CWM V .Although EWMV isolates w ere obtained from w heat , and SBRMV-G and SBRMV-O were from rye , there w ere no obvious molecular differences correlated w ith the host .It seems that two different names have been given inde- pendently to the same virus .Following discussions w ith Dr .Koenig , w e consider that a different name Fig .2 . Symptoms of w heat yellow mosaic virus infected w heat should be chosen that does not im ply restriction to a leaves(a)and w heat field (b). particular geographical region (Europe)or specificity to a particular , and possibly minor , host (rye).The M ost of the cereal viruses w ith the fung al vector name soil-borne cereal mosaic virus seem s to meet have a narrow host range .Under natural conditions, these criteria[ 29, 30] . BaYM V and BaMM V infect barley only , WSSMV , WYM V and CWMV w heat o nly , but CWM V also 1 .2 Disease and host range infects tobacco by mechanical inoculation .OMV and OGSV only infect oats .SBWMV has a w ider host These viruses all cause serious diseases of cereal range and infects w heat and barley .Japanese SB- crops and their biological features extremely depend WMV infects w heat and barley as well as tobacco and upon their property of fungal transmission .After maize .SBCMV naturally infects rye and w heat .Dif- sow ing of cereal seeds in autumn , the resting spores ferences in the genomic sequences of the viruses are of P .graminis , under suitable temperature and no t related to their origins of the natural hosts. moisture in soil , germinate primary zoospores w hich infect root hairs and epidermal cells of the cereal These viruses can survive in the air dried resting crops, and then transmit the viruses into the host spores of P .graminis for many years .The period of cells .It is still not clear whether the viruses as intact survival tested for the follow ing viruses is 9 years for 486 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .6 2005

OGSV , 10 years fo r SBWMV , 10 years fo r BaYMV , 134 reacted only w ith SBWM V Okl-WT and Okl-7 . 5 years for OM V and 5 years for WSSM V . This can be explained by assuming that SCR 134 reacts w ith an epitope that contains Gly-6 near the N- 1 .3 Particle mo rphology terminus of the coat pro tein and this conclusion is supported by the Pepscan data .While monoclonal an- Particles of genus Bymovirus are filamentous tibody SCR 132 reacted with three isolates of SB- w ith typical lengths of 275 —300 nm and 600 — 625 nm and 12 —13 nm in diameter (Fig .3(a)). WMV (SBWMV- Lab 1 , Okl-WT and OKL-7)but The leng ths of a few particles of purified viruses often no t SBWMV-F , w hich differs from the previous exceed 2000 nm because of end-to-end agg regations of three isolates tow ards the C-terminus (Ty r-159 and the particles[ 3, 5] .Particles of genus F urovirus are Thr-160 w ere changed to Ile and Ser , respectively). Thus , it is possible that the SCR 132 epitope contains rod-shaped and their lengths range mainly in 281 — the amino acids Ty r-159 and Thr-160 .The mono- 300 nm and 138 —160 nm w ith the diameter of 20 nm [ 14 , 25] clonal antibody SCR 133 reacted w ith a continuous (Fig .3(b)) . epitope located at one end of the liner coat protein sequence .The epitope was exposed on the surface along the sides of the particles and w as readily removed by try psin- treatment .Since SCR 133 reacted w ith all four isolates , it seems unlikely that it discriminates the SER-6 for Gly-6 change in SBWM V-F and SB- WMV-Lab1 .It is , therefore , probable that it reacts w ith an epitope near the C-terminus , the amino acid residues of w hich are identical in SBWM V Lab1 , SB- Fig .3 . Purified particles of w heat yellow mosaic virus (a) and WMV Okl-W T and SBWM V Okl-7[ 33] . C hinese wheat mosaic virus (b). 1 .5 Deletion mutants 1 .4 Serological affinities In 1994 , we found that repeated passage of SB- M embers of genus Bymovirus have no serologi- WMV by m anual inoculation resulted in deletion of cal relationship with those of genus Furovirus .A- part of SBWM V RNA2 .Deletion w as apparent in the mong the members of genus Bymov irus , BaYMV is population of RNA2 molecules after 11 weeks in pri- serologically related to WSSM V and WYM V , WSS- mary inoculated w heat plants and after 5 passages no M V and WYMV appear to be similar , and OM V and full-leng th RNA2 remained .In earlier passages (pas- BaM MV have no affinities to any members of the sages 1 —4), plants generated several deleted forms of [ 31] genus .Among the members of genus Furovirus , RNA2 w hereas after passage 5 only a stable one re- they are closely serologically related to each other . mained .All naturally deleted forms were cloned and We have produced polyclonal antiserum and mono- the sequences flanking each deletion site were com- clonal antibodies against some members of this genus pared.The results indicated that all deletions oc- for study ing serological relationship between them . curred w ithin the region encoding the coat protein The results indicated that there are some differences read-through domain and the 5′-site of each deletion of epitopes distributed on coat proteins of different laid in the region between genome coordinates 1417 — viruses[ 32] .CWMV and OGSV share the common 1465 while the 3′-sites were very different .After 5th epitopes on the coat proteins at the 30 —40th amino mechanical passage the stable 759 nt deletion became acids, w hile the common epitopes shared by SB- dominant[ 24] .The results also show ed that , in gener- WMV , CWMV , SBCMV and OGSV are located at al , the smaller deletions are not intermediates in the C-terminal half of the coat proteins[ 32, 33] .We have larger deletion process[ 35] . Plants infected by also produced 7 monoclonal antibodies against SB- P .gram inis and maintained at high temperatures WMV .These antibodies have been successfully used (25 —30 ℃)also showed extensive deletions in the to distinguish the different o rigins of SBWM V iso- RT domain of CP-RT gene w ithin 4 —12 weeks .In lates[ 33] .The coat proteins of both SBWMV Lab 1 contrast, plants kept at 17 ℃ over the same period and SBWMV-F differed from SBWM V Okl-WT and contained only full-leng th RNA2 molecules .Our ob- Okl-7 by one amino acid near the N-terminus (Gly to servatio ns confirm that only full-length SBWM V Ser at position 6), and the monoclonal antibody SC R RNA2 is transmitted to w heat roots by viruliferous Prog ress in Natural Science Vol.15 No .6 2005 w ww .tandf.co .uk/journals 487

P .graminis from field soil and there is no intraplant Rong cheng , Luotian , Hanzhong , and Ya' an in Chi- barrier to the movement of deleted forms of RNA2 na , and found that the responses of virus isolates at between roo ts and leaves .Deleted fo rms of SBWMV Yangzhou and Ya' an sites to some selected w heat RNA2 appear to cause more severe symptoms only af- cultivars w ere very different .Fo r instance , some cul- ter mechanical inoculation to y oung , healthy plants . tivars (e .g .Brindur , Italo , Vona)were susceptible These studies may also help elucidate the mechanism at both sites , while o thers (e .g .Hokushin , New ton , of spontaneous RNA deletions[ 34] . Pascal , Tremie and possibly Ernie)appeared to be resistant at both .At Yang zhou site , m any plants of For deletion formation in SBWMV we hypothe- Colosseo , Colfio rito , Platani and Akakomugi w ere in- size that during sy nthesis of plus-strand RNA the vi- fected but these cultivars w ere not , or only slig htly ral replicase pauses and dissociates at minus-strand infected at Ya' an .Conversely , Haruyutaka w as in- structures present at the 5′site .Subsequent reassoci- fected at Ya' an , but no t at Yangzhou[ 10] .The field ation of the replicase occurs at various 3′sites w hich experiments therefore suggest that the tw o isolates of are located randomly (as far as we can tell)in the mi- WYM V differ in their virulence to a range of culti- [ 35] nus-strand RNA .This model is similar to those vars .In Japan , Akakomugi is susceptible to W YMV- proposed to explain the fo rmation of deletions w ithin T but no t to W YMV-H , w hile Haruy utaka is suscep- the genomic RNAs of some animal virus . tible to both strains , but this pattern is no t matched at either of the Chinese sites and their strains are In addition , w e have also identified similar dele- therefore probably different from those reported in tion mutants from the mechanically inoculated OGSV Japan[ 39] . and CWMV in w hich the deletions occur w ith RT domain of RNA2 , but that of the mechanically inoculat- 2 Polymyxa graminis ed OMV occurs w ithin P2 gene as described above .It seems that the deleted form s of all viruses are not 2 .1 Life cycle transmissible by fungus[ 12 , 22] . In 1939 , Ledingham first discovered Polymy xa 1 .6 Pathogenicity gram inis on wheat in Canada .Since then , he spent 9 years to observe mo rphology and life cycle of this fun- Disease control relies almost exclusively on the gus[ 40] .In the last decade , we have sy stem atically deployment of resistant crop cultivars .Fo r many of studied the development of this fungal vector by elec- the fungus-transmitted cereal viruses , little is know n tron microscopy[ 41—44] .The life cy cle of the fungal about the interactions betw een virus strains and host vector co nsists of biflagellate zoospores (Fig .4(a)), geno types or the viral determinants of virulence .The multinucleate plasmodia , zoosporang ia and thick- only significant exception is for the bymoviruses of w alled resting spores in clusters or cystosori (Fig .4 barley , BaMM V and BaYM V . Seven different (b)).Thick-w alled resting spo res are fo rmed in plant strains of BaYMV (in four g roups)have been identi- roots and rem ain in the soil when the roo ts decay . fied in Japan based on the response of different culti- The prim ary zoospores , produced by resting spo re vars.In Europe , tw o strains are recog nized on the germination , swim to host root hairs or epidermal basis of the response of cultivars carrying the ry m4 surfaces w hen they encyst , a process that involves resistance gene and the resistance-breaking strain , w ithdraw al of the flagella , adhesion to the host w all usually named BaYM V-2 , is becoming increasingly and secretion of a thin cyst w all .The zoospore proto- important .In the 1990s , we have examined the re- plast is injected into the host cell , w here it divides in- sponses of some selected barley cultivars to BaYMV at to a multinucleate plasmodium (spo rangia plasmodi- 10 sites distributed in different areas of the disease in um)and develops into zoosporangium .This sac-like China and found that at least 6 strains probably pre- structure liberates secondary zoospores (which do not sent[ 36] .To rapidly detect and diagnose these strains , differ morphologically from the prim ary zoospo res) w e have developed some molecular techniques includ- into soil w ater o r perhaps to penetrate deeper into the ing RT-PC R-single-strand co nformation polymor- host root , thus continuing the infection cycle .The phism (SSCP)and restriction mapping[ 9 , 37, 38] . factors leading to resting spore formation are no t un- derstood :both ex ternal facto rs (e .g .host nutrition , For WYMV , w e carried out field experiments at stress)and internal ones (kary og amy)have been in- 7 sites including Yangzhou , Huang chuan , Yantai , voked .In early stages of development , there is no ap- 488 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .6 2005 parent difference between sporogenic and spo rangial isolates studied in detail have originated from barley , plasmodia .The first evidence of sporogenesis is the and they multiplied best on barley and usually less development of synaptonemal complexes and this is well on w heat ;isolates from w heat may have multi- followed by meio tic divisio n and formation of thick- plied better in w heat than did the barley isolates and w alled resting spores .How ever , when and w here cell none of these isolates multiplied appreciably in fusion and karyogamy occur rem ains a mystery in the oats[ 49] .Further wo rk is needed to determine the de- life cy cle of the fungus . gree of specialization to different hosts and to ex plo re further the susceptibility of w eed hosts to the viruses that the fung al vector transmits .In addition , it is possible to determine the absolute numbers of cy s- tosori and the germination ratio of the resting spores of P .graminis .Our studies show ed that the fungus has a g reat potential of infection .In baiting test using infested soil, the dilution endpoints for detecting WYM V on w heat roo ts were 1/625 —1/15625 , and fo r resting spo res of the fungus vector , P .graminis , 1/3125 —1/15625[ 45] .Electron microscopic ob- servatio ns showed that resting spo res of the fungus collected from roots of wheat after harvest were mostly empty , usually associated with many bacteria , sug- gesting that germination had occurred in situ[ 43, 44] .

2 .3 Relationship between viruses and the fungal vector

Fig .4 . Polymy xa graminis:(a)Zoospore;(b)resting spores; Since the 1960s , scientists in the international (c)barley mild mosaic virus particles inside zoospore and (d)barley society of plant pathology have been co ntinuing to ex- yellow mosaic virus particles inside resting spore .(a)and (b)are plore the relationships between the fungal vectors and provided by M .J.Adams. plant viruses they transmit although the progress is 2 .2 Epidemiology rather slow .Virus transmission occurs by zoospore , but in one of two different w ay s .Those viruses trans- We have studied the effects of environmental fac- mitted by Oplidium brassicae are acquired from soil tors on the development of the fungal vector , w ater on the out surface mem brane of zoospores .The P .graminis in the g reenhouse using irrigated sand virus particles appear to enter the zoospore through cultures .The isolates collected from the temperate re- the infection canal but they do not enter the resting gions were able to g row and infect barley roots over a spore during its formation .By contrast , all viruses w ide range of temperature , but the fastest develop- transmitted by Plasmodiophoromycates including P . ment occurred at 17 —20 ℃ and it only took about 2 gram inis are acquired from plant cells and enter the w eeks from inoculation w ith resting spo res to produce resting spores of vectors and released w ith zoospores secondary zoospo res .The isolates from tropical re- on germination .M embers of genera Bymovirus and gions , w hich transmit peanut clump virus , had a Furovirus belong to the second type of transmission , higher optimum temperature of 27 —30 ℃. but how and w hen P .graminis acquires viruses, and how viruses enter the host plant cell cytoplasm P .gram inis has been detected in roots of culti- are unknow n .However , it is likely that these pro- vated w heat , barley , rice , oats , rye , m aize and cesses are taking place either w hen zoospore pene- so rghum .It has also been detected in the roo ts of trates the host cells and transferes their contents into Bermuda grass and creeping bent grass as w ell as vari- the host cell cytoplasm , or at the spo rogenic plas- ous temperate Agrostis , Dactylis , Festuca , Poa and modia stage of P .graminis development when there Phleum species.This is w orrying because all these is only a thin membrane boundary separating the plas- w ild g rass species might serve as reservoirs fo r modioplorid from the host cell cy toplasm[ 49] .Ac- P .graminis , the ex tent to w hich different isolates quired viruses are thought to be carried inside the fun- can infect all these hosts is largely unknow n[ 49] .M ost gus spo res and cannot be removed from zoospores by Prog ress in Natural Science Vol.15 No .6 2005 w ww .tandf.co .uk/journals 489 w ashing or treatment of antiserum , or inactivated by 4 Prospects application of NaOH and HCl.Using immunogold labeling electron microscopy technique in 1991 , w e ob- Research prog ress in understanding and control- served M aM MV particles inside a very sm all portion ling the virus diseases has been rather slow because of (c .1 %)of zoospores and zoosporangial plasmodia of the difficulties of studying w ith an oblig ate , root in- P .graminis (Fig .4(c))although it is proved that it fecting fungal vecto r .However , these challenges [ 47, 48] must be met if many of the intrig uing questions about transmits the virus efficiently .Seven y ears lat- the diseases are addressed .The follow ing areas are of er , we also found BaYM V-like particles inside a few resting spores of the fungus (Fig .4(d))[ 44] .This is particular interest and relev ance :Molecular studies are required to clarify various aspects of the taxonomy the first evidence in which immunogold labeling has and phy logeny of the plasmodiophorids ;the biological been used to confirm the identity of a virus in its fun- differences, such as host range , transmissibility of g al vector . different viruses , and difference between the various 3 Disease resistance isolates of P .gram inis need to be further studied ; mechanisms of germination of the resting spores and Although P .graminis is an oblig ate parasite of recog nizing hosts of the zoospores , molecular mecha- cereal roots , it is not considered a pathogen because it nism of requiring and transmission of the viruses by does no t cause any disease , and does not seem to re- the fungal vector , molecular mechanism of viral duce crop yield .However , it transmits several plant patholognicity , mechanism of resistance of cereal viruses that do cause serious diseases of cereal crops . crops to the viruses , and mechanism of resistance Since virus-containing resting spores of P .gram inis breaking of the cereals to the viruses are all important persist in soil and crop debris for decades , cultural issues to be addressed .Answ ering these questions has practices such as crop rotations or delayed sow ing fo r g reat scientific value to understand the interactio ns a- virus control are of little value , w hilst chemical con- mong the fungal vecto r , viruses and host plants , and trol methods are unacceptable for ecological and eco- to establish new systems of disease control. nomic reasons .Therefore , application of resistant ce- References real varieties offers the only practical and economically friendly measure of control .In the past decade , we 1 Rao A .S .and Brakke M .K .Relation of soil-borne w heat mosaic virus and its fungal vector , Polymy xa graminis .Phytopathology , carried out a project of screening resistant genoty pes 1969 , 59 :581 —587 . imm une to bymoviruses and furoviruses from a large 2 Chen J.P .Occu rrence of fungal transmitted w heat mosaic viruses number of germsplasm s of w heat and barley and in China .Annals of Applied Biology , 1993 , 123 :56 —61 . 3 Chen J.P ., Ruan Y.L .and Dong M .J.Properties of barley yel- found 4 Japanese local barley varieties (Chosen , low mosaic virus and production of its an tiserum .Zhejiang Agricul- Hagnne Mugi , Iw ate Mensumy 2 and Mokusekko 3) tural Sciences, 1989 , 5 :239 —241 . and a European one (Energy)im mune to bymoviruses 4 Chen J.P ., Ruan Y.L .and Hong J.Cytoplasmic inclusions and organelle alternations in barley infected by barley yellow mosaic of barley from China , Japan and Europe , and these virus (BaYMV).Virologica Sinica, 1990 , 5 :207 —213 . resistant resources have been used in barley breeding 5 Chen J.P .and Ruan Y .L .Purification , serology and other prop- prog rammes[ 18 , 21] .In addition , w e have also identi- erties of w heat yellow mosaic virus (WYMV).Virologica Sinica , 1990 , 3(2):193 —200 . fied 47 fo reign w heat varieties (Chisholm , Victory 6 Chen J .P ., S hi C ., Zhu F .T .et al.First report of the occur- etc .)resistant to WYMV and 4 (Karl , Larned , Pan- rence of barley mild m osaic virus (BaMMV)in China.Virologica das and H aw k)resistant to CWMV in China[ 26] .Ce- Sinica, 1993 , 8(1):107 —110 . 7 Chen J., Chen J .P., Du J.Z .et al.Sequence diversity in the real crop cultivars w ith good resistance to one or more coat protein coding region of w heat yellow mosaic bymovirus iso- of fungus-transmitted viruses are now commercially lates from China.Journal of Phytopathology , 2000 , 148(9 —10): available , but germ plasms of w heat and barley w hich 515 —521 . 8 Zheng T ., Cheng Y ., Chen J.P.et al.Confirmation of barley are immune to P .graminis have not been available , mild mosaic virus (BaM MV)in China and the nucleotide sequence o r need to be further confirmed .Nevertheless , resis- of its coat protein gene .Journal of Phy topathology , 1998 , 147 tance to the fung al vector could provide the oppo rtu- (4):229 —234 . 9 Chen J., S hi N .N., Cheng Y.et al.Sequence analysis of barley nity to co ntrol all the viruses w hich the fungal vecto r yellow mosaic virus from China.Virus Research , 1999 , 64(1): transmits simultaneously and is therefore an attractive 13 —21 . idea[ 49] .Some germplasm of Hordeum bulbosum ap- 10 Chen J., Chen J.P ., Yang J.P .et al.Differences of cultivar response and complete sequence analysis of two strains of w heat yel- pears to be resistant to P .gram inis and might pro- low mosaic bymovirus in C hina .Plant Pathology , 2000 , 49(3): [ 50] vide a source of resistance for barley breeding . 370 —374 . 490 w ww .tandf.co .uk/journals Prog ress in Natural Science Vol .15 No .6 2005

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