K.R. Cui1, J.M. Krupinsky2, Q. Dai3, E. Arseniuk4 and P.P. Ueng5
1
State Key Lab o ra tory of Arid Agroecology, Lanzhou Uni ver sity, Lanzhou, Gansu, Peo ple’s Re pub lic of China; 2North ern Great Plains Re search Lab., USDA-ARS, Mandan, ND, USA; 3De part ment of Bi ol ogy, Shandong Uni ver sity, Jinan, Shandong, Peo ple’s Re pub lic of China; 4Plant Breed ing and Ac cli ma ti za tion In sti
-tute, B³onie, Po land; 5Mo lec u lar Plant Pa thol ogy Lab., USDA-ARS, BARC-West, Beltsville, MD, USA
CELL WALL-DEGRADING ENZYMES AND AGGRESSIVENESS IN STAGONOSPORA NODORUM
ABSTRACT
Stagonospora nodorum pro duces cell wall de grad ing en zymes when grown in cul ture me dia con tain ing cell wall com po nents. The patho gen grew as well on min i mal agar plates con tain ing cel lu lose, xylan and pec tin as glu -cose, ex cept hav ing sparser mycelia. Four cell wall-de grad ing en zymes, cellulase, xylanase, pectinase and b1,3glucanase were coordinately in duced in cul ture fil trates grow ing on xyaln and cel lu lose as sub strates. An ag -gres sive iso late (sn26-1) se creted more cell wall-de grad ing en zymes than the oth ers. Based on isoelectric fo cus ing pro files, six to seven xylanase isozymes were in duced by cel lu lose and xylan. No dif fer ence was found in the high (sn26-1) and low (9074) ag gres sive iso lates. Ad di tion of cell wall-de grad ing en zyme mix tures, not high xylanase alone, to a spore sus pen sion of a low ag gres sive iso late (9074) caused a lim ited in crease in tis sue ne cro sis. We con -clude that the cell wall de grad ing en zymes play a role in early pen e tra tion of the host by the fun gus, but they are not im por tant elicitors for dis ease de vel op ment.
Key words: ag gres sive ness, b-1,3-glucanase, cellulase, isozymes, isoelectric fo cus ing, pectinase, se cre tion, Stagonospora nodorum, xylanase
INTRODUCTION
The fil a men tous fungi uti lize plant ma te ri als as the source of car bon and en ergy through the ac tion of many cell wallde grad ing en zymes. Se cre tion of cell wallde -grad ing en zymes such as xylanases (1,4-b-D-xylan xylanohydrolase; EC 3.2.1.8) was de tected in many plant spe cies in fected by leaf patho gens (Coo per et al. 1988, Waksman and Keon 1989, Mendgen et al. 1996, Mbwaga et al. 1997, Xu and Mendgen 1997, Schmidt and Wolf 1999). It was spec u lated that cell wallde grad -ing en zymes would mac er ate plant cell walls and fa cil i tate fun gal pen e tra tion and in fec tion. Many fungi in cit ing de struc tive soil-borne dis eases in plants also show an adap tive high level of pro duc tion of these en zymes in cell wall me dia and in -fected tis sues (Johansson 1988, Southerton et al. 1993, Dori et al. 1995, Bidochka
et al. 1999, Großwindhager et al. 1999). In ad di tion to en zy matic ac tiv ity, fun gal
xylanases also act as proteinaceous elicitors of de fense re sponse re ac tions in di cots.
Communicated by Andrzej Anio³
P L A N T B R E E D I N G A N D S E E D S C I E N C E
Volume 51 2005
Elic i ta tion of eth yl ene biosynthesis, pathogenesisre lated pro teins and tis sue ne -cro sis was as so ci ated with this group of en zymes (Bailey et al. 1990, Lotan and Fluhr 1990, Sharon et al. 1993, Enkerli et al. 1999, Furman-Matarasso et al. 1999). In re cent stud ies, there were re ports that cell wall de grad ing en zymes were se creted by Stagonospora nodorum, the caus ing agent of glume blotch dis ease in wheat (Magro 1984, Lehtinen 1993, Lalaoui et al. 2000). Xylanase ac tiv ity was highly de tected in an ag gres sive iso late of S. nodorum, and was con sid ered to play a cer -tain role in pathogenesis (Lalaoui et al. 2000). In this study, we com pared the xylanase and other cell wall-de grad ing en zyme pro duc tion in some low and highly ag gres sive iso lates of S. nodorum and eval u ated the re la tion of these en zymes to fun gal ag gres sive ness.
MATERIALS AND METHODS Fun gal iso lates and growth me dia
Eight iso lates of S. nodorum in clud ing four highly ag gres sive and the four low ag gres sive were stud ied (Ta ble 1). Two low ag gres sive iso lates, 9074 and 9076, were iso lated from wheat and pe ren nial grass (Krupinsky 1997a, 1997b). They caused mild symp toms on wheat and triticale (Arseniuk et al. 1999). Iso lates 64-1 and 64-4 were prog eny of a sex ual cross be tween iso lates sn26-1 and sn48-1. These two ascosporede rived cul tures had low infectivity due to poor sporulation (un pub -lished data). Fun gal cul tures were main tained on V8 juice agar (18% V8 juice, 0.2% cal cium car bon ate and 1.5% agar) plates at 22°oC (Stevens 1974).
Cell growth and en zyme as says
Fun gal cul tures were first trans ferred three times as agar blocks and main tained on yeast ni tro gen base (YNB, Difco Lab, De troit, MI) agar me dia con tain ing var i -ous poly sac cha rides such as cel lu lose, xylan, and pec tin as the sole car bon sources (Sigma, St.Louis, MO). The mycelial growth on glu cose, cel lu lose, xylan and pec
-Ta ble 1.
Iso lates of Stagonospora nodurum used for cell-wall-de grad ing en zymes anal y sis.
Isolates Original hosts Geographic location Aggressiveness on wheat b
sn26-1 Winter wheat Rzeszów, Poland High
sn48-1 Winter rye Lower Silesia, Poland High
9506 Barley Morton, ND High
8408 Wheat Morton, ND High
9074 Wheat Gallatin, MT Low
9076 Wheat Richland, MT Low
64-1a - - Low
64-4a - - Low
a
Prog eny of a sex ual cross sn26-1xsn48-1. bIn a 0 – 9 scale, the per cent age of ne cro sis >4.0 is ‘high’ in ag gres sive -ness. If <2.0, it is ‘low’ in ag gres sive ness (Saari and Prescott 1975).
tin sub strates were de ter mined by in oc u lat ing the re spec tive agar plates with in di -vid ual agar blocks con tain ing the mycelia, main tain ing the plates at 26+2°C un der 24hr flu o res cent lights and mea sur ing the col ony di am e ter. The ex per i ment was re -peated three times, each with three plates. For en zyme as says, the agar blocks from YNBxylan and YNBcel lu lose plates were in oc u lated to YNB liq uid me dia con -tain ing xylan and cel lu lose, re spec tively and grown at 26°C with con stant shak ing (200 rpm). The mycelial mass was har vested by centrifugation. The en zy matic ac -tiv ity in the supernatants was de ter mined by mea sur ing the amount of re duc ing sug ars re leased from the sub strates with glu cose as the stan dard (Miller 1959). Cel -lu lose (2%, W/v, pH 5.0), oat spelt xylan (2%, w/v, pH 5.0), polygalacturonic acid (0.8%, w/v, pH 4.0) and laminarin (0.5%, w/v, pH 5.0) were used as as say sub strates to de tect cellulase, xylanase, pectinase and b1,3glucanase ac tiv i ties, re -spec tively (Lehtinen, 1993). The pro tein con cen tra tion was de ter mined with a BCA Pro tein As say Kit (Pierce, Rockford, IL) us ing bo vine se rum al bu min as the stan dard.
Isoelectric fo cus ing (IEF)
The IEF pro files of xylanase isoenzymes from sn261 and 9074 grown on cel lu -lose as sub strate were com pared. An acrylamide gel con tain ing 8.25 g urea, 2ml of 30% acrylamide/1.8% bis, 0.75ml ampholine (pH3.5-10.0), 0.2ml 2%(v/v) TritonX-100, 6.0 ml wa ter, 5 µl TEMED and 35 µl 10% am mo nium per sul fate was pre pared. The extracellular pro teins in cul ture fil trates were pre cip i tated with 0-95% sat u rated am mo nium sul fate, re sus pend ed in 0.1M phos phate buffer (pH6.0), and de salted and con cen trated by cen trif u gal fil tra tion (Biomax5 mem -brane with a 5,000 nom i nal molcular weight limit cut-off, Millipore, Bed ford, MA). The par tially pu ri fied pro tein sam ple with a 500 µmol/hr xylanase ac tiv ity was mixed with IEF sam ple dye and run by elec tro pho re sis (Hoefer SE260 Mighty Small II, Amersham Pharmacia Biotech Inc., Piscataway, NJ). A broad isoelectric point (pI) kit (pH 3.5-9.3) was used for pI de ter mi na tion (Amersham Pharmacia Biotech Inc.). Phos pho ric acid 0.085% (v/v) and 0.02M NaOH were used as ano -lyte and catho-lyte buff ers, re spec tively. The gel was run at 300 volts for 20 h and 1000 volts for 7 h con tin u ously with cir cu lated cool ing wa ter (15°C). Xylanase ac -tiv ity in IEF gel was de tected fol low ing the method of Mac Ken zie and Wil liams (1984). The electrofocused gel was soaked in a 0.2M so dium ci trate-HCl (pH 4.5) buffer at room tem per a ture for 30 min. The gel was then placed in con tact with sub -strate gel con tain ing 0.2M so dium ci trate-HCl (pH 4.5), 1.0% agarose and 0.75% 4-O-methyl-D-glucurono-D-xylan-remazol bril liant blue R (Sigma, St. Louis, MO) and wrapped in poly vi nyl film. Af ter in cu ba tion at 55oC for 90 min, the gel was fixed with eth a nol/so dium ci trate-HCl buffer (pH 4.5) (2/1 v/v) for 48 h. The xylanase isozyme bands were dis played as clear bands in a blue back ground of sub strate gel. For pro tein stain ing, the gel was im mersed in GELCODE blue stain re -agent (Pierce, Rockford, IL) for 48 h.
In oc u la tion tests.
To de ter mine xylanase and cellulase ac tiv i ties in dis eased leaves, S. nodorum in -clud ing two highly ag gres sive (9506 and 8408) and two low ag gres sive (9074 and
9076) iso lates was in oc u lated on wheat. The inocula were pre pared by grow ing the cul tures on V-8 juice agar at 21oC un der con tin u ous cool white flu o res cent light for 16 days (Stevens 1974). Pycnidiospores (2-3x106/ml) were sus pended in ster ile deionized wa ter with Tween20. The 10-day-old wheat seed lings of ‘Fortuna’ (CI 13596, sus cep ti ble) and ‘Red Chief’ (CI 12109, re sis tant) were sprayed with 3 ml of pycnidiospore sus pen sion. The con trol plants were sprayed with wa ter only. Leaves were har vested and frozen with liq uid ni tro gen at 0, 3, 6 and 9 days af ter in -oc u la tion. The leaf ex trac tion fol lowed the pro ce dure de scribed ear lier (Seevers et
al. 1971). The leaf ex tracts were pre cip i tated with am mo nium sul fate, de salted and
con cen trated by cen trif u gal fil tra tion. En zy matic ac tiv i ties and pro tein con cen tra -tion in the par tially pu ri fied leaf ex tracts were de ter mined.
To de ter mine the ef fects of cell wall-de grad ing en zymes on ini tial in fec tion by S.
nodorum, par tially pu ri fied cul ture fil trates were added to spore sus pen sions in
a de tached leaf test (Baker and Smith 1978). The 5 cm-long pri mary leaf seg ments were cut from 8dayold seed lings of wheat ‘Olaf’ (CI15930, mod er ately sus cep ti -ble), and placed in a square plas tic Petri plate (100 × 15mm) con tain ing 0.015% benzimidazole and 0.5% agar. S. nodorum iso lates 9506 and 9074 were used as inocula. A 5µl drop let of spore sus pen sion (8,000 spores) from the low ag gres sive iso late 9074 con tain ing 10 mM phos phate buffer (pH 6.0) and var i ous en zyme con -cen tra tions was added to the -cen ter of a de tached leaf. Five con -cen tra tions of high xylanase/cellulase en zyme ra tio mix ture (4k/15, 8k/30, 12k/45, 16k/60 and 20k/75 nmol/h) from sn261 grown on xylan and low xylanase/cellulase en zyme ra tio mix -ture (4k/2.8k, 8k/5.6k, 12k/8.4k, 16k/11.2k and 20k/14k nmol/h) from sn26-1 grown on cel lu lose were used. The spore sus pen sion of highly ag gres sive iso late 9506 was used as the con trol. Ten leaf seg ments rep li cates were used for each treat -ment. Nine days af ter in oc u la tion, over all se ver ity of in fec tion (as per cent ne cro sis) on the de tached leaf sec tions was rated by di rect mea sure ments of le sion size (Shaner and Buechley 1994).
RESULTS
Cell growth and en zyme as says
All S. nodorum iso lates ex cept 9076 adapted to cor re spond ing cell wall com po -nents and grew well on YNB agar me dia con tain ing the cell wall com po -nents as ma jor car bon and en ergy source (Fig. 1). The mycelia grew well in glu cose, cel lu -lose, and xylan, but poorly in pec tin (for 8408 and 9506-1 iso lates not shown). How ever, the mycelial growth ap peared to be denser in glu cose than the other three cell wall com po nents.
It was dem on strated that cell wall-de grad ing en zymes, such as cellulase, xylanase, pectinase and b-1,3-glucanase can be coordinately pro duced us ing ei ther xylan or cel lu lose as sub strates (Fig. 2). Nev er the less, xylanase ac tiv i ties were much higher in two highly ag gres sive iso lates (sn261 and 9506) and one low ag -gres sive iso late (64-4) grown on xylan than those on cel lu lose. The in duc tion of xylanases by cel lu lose and xylan was prob a bly equal in three other low ag gres sive iso lates (9074, 9076 and 64-1) (Fig. B). When the S. nodorum cul tures were grown on dif fer ent sub strates, var i ous pro tein con cen tra tions were de tected in the liq uid
me dia. The av er age amount of pro teins (µg/ml) se creted into cul ture me dia by six
S. nodorum iso lates was 3,059 for glu cose, 467 for pec tin, 432 for xylan, and 52 for
cel lu lose. Even though the cul tures grown on cel lu lose se creted the least amount of pro teins, they in duced si mul ta neously a rea son able amount of cell wall-de grad ing en zymes (Fig. 2).
A B
C D
Fig. 1. Mycelial growth of Stagonospora nodorum iso lates dif fer ing in ag gres sive ness on var i ous cell wall com -po nents. Yeast ni tro gen base agar plates con tain ing 0.5% glu cose (GLU) (A), cel lu lose (CEL) (B), xylan (XYL)
(C), and pec tin (PEC) (D), re spec tively, were used. The bars in di cate the stan dard de vi a tion
A. Xylan B. Cellulose 0 20 40 60 80 100 P G C X E n zy m a tic A ct iv iti e s (n m o l/h r/ u g p ro te in ) sn26-1 9506 9074 9076 64-1 64-4 0 10 20 30 40 50 P G C X E n zy m a tic A ct iv iti e s (n m o l/h ru g p ro te in ) sn26-1 9506 9074 9076 64-1 64-4
Fig 2. Se cre tion of cell wall-de grad ing en zymes by Stagonospora nodorum iso lates dif fer ing in ag gres sive ness on ei ther xylan (A) or cel lu lose (B). Four en zy matic ac tiv i ties (nmol/hr/µg pro tein) in clud ing xylanase (X),
Isoelectric fo cus ing
Based on isoelectric fo cus ing (IEF) pro files, the same six to seven xylanase isozymes could be de tected in highly ag gres sive (sn26-1) and low ag gres sive (9074) iso lates (Fig. 3). Two of the isozymes were ba sic pI and the oth ers acidic.
In oc u la tion tests
No sig nif i cant xylanase and cellulase ac tiv ity could be de tected in ‘Fortuna’ and ‘Red Chief’ leaf tis sue ex tracts in fected with two highly ag gres sive and two low ag gres sive S. nodorum iso lates (data not shown). In the de tached leaf test, no sig nif i -cant dif fer ence was found in pycnidiospore inocula of low ag gres sive iso late 9074 con tain ing high xylanase/cellulase en zyme ra tio mix tures (Ta ble 2). Nev er the less, at the 10% sig nif i cance level dif fer ences were found in two high low xylanase/cellulase en zyme ra tio mix tures (16/11.2 and 20/14) as com pared to low
Fig. 3. Isoelectric fo cus ing (IEF) pro files of xylanase isozymes from two Stagonospora nodorum iso lates dif fer -ing in ag gres sive ness. Par tially pu ri fied en zyme ex tracts from 9074 (1) and sn26-1 (2) were used
Ta ble 2
Com par i son of per cent ne cro sis on the de tached leaf seg ments in fected with pycnidiospores of low ag gres sive Stagonospora nodorum iso late 9074 con tain ing var i ous xylanase/cellulase
con cen tra tions from the high ag gres sive iso late 9506.
Treatment with high (xylanase/cellulase)a enzyme ratio mixture
Mean difference of percent
necrosis (xylanase/cellulase)Treatment with lowa
enzyme ratio mixture
Mean difference of percent necrosis 9074 9506 9074 9506 9074 (-) - 14** 9074 (-) - 8** 9074 (4/0.015) 1 13** 9074 (4/2.8) 0 8** 9074 (8/0.030) 0 14** 9074 (8/5.6) 1 7** 9074 (12/0.045) 1 13** 9074 (12/8.4) 1 7** 9074 (16/0.060) 2 12** 9074 (16/11.2) 3D 5* 9074 (20/0.075) 0 14** 9074 (20/14.0) 3D 5* a
The unit of en zy matic ac tiv ity is µmol/h.
D
in di cates the dif fer ence be tween two en zyme-sup ple mented sam ples and the low ag gres sive iso late 9074 (con trol) are sig nif i cant at 10% level in the t-test.
*
and ** in di cate the dif fer ences be tween highly ag gres sive iso late 9506 (con trol) and low ag gres sive iso late 9074 treated with var i ous en zyme mix tures are sig nif i cant at 5% and 1% lev els, re spec tively
ag gres sive con trol iso late 9074. The dif fer ences be tween the highly ag gres sive con trol iso late 9506 and the low ag gres sive iso late 9074 with var i ous en zyme ra tio mix tures were sig nif i cant at the 1% and 5% lev els (Ta ble 2).
DISCUSSION
Cellulases and many other cell wall-de grad ing en zymes, like xylanase and polygalacturonase, were coordinately in duced in cul tures of soil-borne fun gal patho gens grown on wheat straw and other cel lu lose prod ucts, even though the max i mal amounts of these en zymes would be pro duced in me dia con tain ing the cor re spond ing sub strates (Lynch et al. 1981, Bahkali 1995). One pos si ble in duc -tion of these cell wall-de grad ing en zymes in cel lu lose grown cul tures was due to the pres ence of sub strate con tam i nants in cel lu lose prod ucts (Lynch et al. 1981, Royer et al. 1992). The other pos si bil ity, the pro duc tion of a num ber of cell wallde grad ing en zymes is coreg u lated with the pres ence of a sin gle sub strate, ei ther cel lu lose or xylan. In some cases, the pro duc tion of cell wall de grad ing en -zymes, such as xylanase and pectinase in Rhizoctonia solani, mannanase in
Sclerotium spe cies, arabanase and laminarinase in Rhizoctonia cerealis, Fusarium culmorum and Pseudocercosporella herpotrichoides, and pectinases in Sclerotinia sclerotiorum is con sti tu tive (Coo per et al. 1988, Robson et al. 1989, Riou et al.
1991, Großwindhager et al. 1999). In our study, none of four cell wall de grad ing en zymes in S. nodorum were de tected in glu cose me dia and were con sti tu tively ex -pressed. It took two days for S. nodorum cul tures grown on YNB-glu cose agar to pro duce xylanases for xylan hy dro ly sis (un pub lished data). We are not sure if the pH of cul ture me dia or other en vi ron ment fac tors would af fect cell wall-de grad ing en zyme se cre tion in this fun gus (Bailey et al. 1993).
Mi cro bial xylanases be long ing to the class of glycosyl hy dro las es are as signed to two fam i lies: fam ily 10 con tains en zymes with a mo lec u lar weight over 30kD and an acidic pI, and fam ily 11 com prises those with a mo lec u lar weight smaller than 30kD and a ba sic pI (Henrissat and Bairoch 1993, Biely et al. 1997). The num ber of xylanase isozymes de tected in S. nodorum cul ture fil trates var ied. Magro (1984) re -ported a sin gle xylanase with pI 5.2 was iden ti fied in liq uid cul tures and dis eased leaf tis sues. Nev er the less, up to 13 acidic xylanase isozymes (pI range 3.6 - 6.2) were in duced with oat spelt xylan and wheat cell wall sub strates (Lehtinen 1993). In this study, us ing a broad range ampholine (pH 3.5-10.0) in IEF, 6-7 xylanase isozymes were found in S. nodorum cul ture fil trates grown in xylan and cel lu lose sub strates. How ever, two low ba sic or near neu tral pI xylanase isozymes were also found. Xylanases with high ba sic pI val ues were re ported in soil-borne fungi and sug gested to be im por tant in pathogenesis (An der son et al. 1993, Southerton et al. 1993).
In gen eral, pro duc tion of cell wall-de grad ing en zymes by fun gal patho gens in leaf tis sues is un der catabolite re pres sion. Ex pres sion of a pro tein kinase gene,
SNF1, in fun gal cells to re lease from catabolite re pres sion is nec es sary for ac ti vat
-ing the cell wall-de grad -ing en zymes, de grad -ing and me tab o liz -ing the plant cell wall com po nents and be com ing vir u lent to the host plant (Tonukari et al. 2000). Ex pres sion of two xylanase genes was proven with cy to log i cal and mo lec u lar tech
-niques in Claviceps purpurea-in fected rye ovar ian tis sues (Giesbert et al. 1998). In
S. nodorum, ac tiv i ties of three cell wall-de grad ing en zymes, cellulase, pectinase
and xylanase, in the dis eased tis sue ex tracts were re ported (Magro 1984). How ever, ac tiv i ties of xylanase and cellulase in S. nodorum in fected tis sue ex tracts were not sig nif i cant in our study.
Stud ies on the role of cell wallde grad ing en zymes in plant pathogenesis are lim -ited and con tro ver sial. De tec tion of these en zymes in the in fected tis sues sug gested that se cre tion by fun gal patho gens would fa cil i tate hyphal pen e tra tion and in crease patho ge nic ity (Netzr et al. 1979, Coo per 1989, Esquerré-Tugayé et al. 1989, Waksman and Keon 1989, Mendgen et al. 1996, Mbwaga et al. 1997, Xu and Mendgen 1997, Schmidt and Wolf 1999). How ever, in the study of ex pres sion of a sin gle gene prod uct, such as cellulase (Sposato et al. 1995), pectinases (Walton 1994) and cutinase (Kolattukuday et al. 1995, Ol i ver and Osbourn 1995), it is dif fi -cult to de ter mine their in volve ment in fun gal patho ge nic ity. The func tion of cell wall-de grad ing en zymes, par tic u larly xylanases, in S. nodorum ag gres sive ness is not clear (Lalaoui et al. 2000). High xylanase pro duc ing iso late, sn261, did not in -cite se verer symp toms than the other highly ag gres sive iso lates in ce re als (Arseniuk et al. 1999). In this study, a lim ited im pact by cell wallde grad ing en -zymes on leaf ne cro sis in di cates they may fa cil i tate early fun gal pen e tra tion but not ag gres sive ness. Ag gres sive ness in S. nodorum ap pears to be con trolled by other ge -netic el e ment(s).
ACKNOWLEDGMENTS
The au thors thank B.M. Cunfer of Uni ver sity of Geor gia for re view ing the manu -script. This work is par tially sup ported by the USDA-ARS CRIS pro ject.
REFERENCES
An der son J.D., Bailey B.A., Tay lor R., Sharon A., Avni A., Mattoo A.K., Fuchs Y. (1993) Fun gal xylanase elic its eth yl ene biosynthesis and other de fense re sponses in to bacco. in Pech J.C., Latché A., Balagué C. (eds.). ‘Cel lu lar and mo lec u lar as pects of the plant hor mone eth yl ene.’ Vol 16, Kluwer Ac a demic Pub lish ers, Dordrecht, The Neth er lands, p.197-204
Arseniuk E., Tsang H.S., Krupinsky J.M., Ueng P.P. (1999) Char ac ter iza tion of less ag gres sive Stagonospora nodorum iso lates from wheat. In Septoria and Stagonospora dis eases of ce re als: a com -pi la tion of global re search. (van Ginkel, M., McNab, A., and Kru-pinsky, J., eds.) Mex ico, D.F.: CIMMYT, 186pp, p.83-84
Bahkali A.H (1995) Pro duc tion of cellulase, xylanase and polygalacturonase by Verti cilli um tricorpus on dif fer ent sub strates. Bioresource Technol. 51: 171-174
Bailey B.A., Dean J.F.D., An der son J.D. 1990 An eth yl ene biosynthesisin duc ing endoxylanase elic its elec -tro lyte leak age and ne cro sis in Nicotiana tabacum cv. Xanthi leaves. Plant Physiol. 94: 1849-1854 Bailey M.J., Buchert J., Viikari L. 1993 Ef fect of pH on pro duc tion of xylanase by Trichoderma reesei on
xylan- and cel lu lose-based me dia. Appl. Microbiol. Biotechnol. 40: 224-229
Baker E.A., Smith I.M. 1978 De vel op ment of re sis tant and sus cep ti ble re ac tions in wheat on in oc u la tion with Septoria nodorum. Trans. Br. Mycol. Soc. 71: 475-482
Bidochka M.J., Burke S., Ng L. 1999 Extracellular hydrolytic en zymes in the fun gal ge nus Verti cilli um: ad -ap ta tions for pathogenesis. Can. J. Microbiol. 45: 856-864
Biely P., Vršanská M., Tenkanen M., Kluepfel D. 1997 Endoâ1,4xylanase fam i lies: dif fer ences in cat a -lytic prop er ties. J. Biotech. 57: 151-166
Coo per RM. 1989 Host cell wall loos en ing and sep a ra tion by plant patho gens. In NATO ASI se ries H35: 165-178
Coo per R.M., Longman D., Camp bell A., Henry M., Lees P.E. 1988 En zy mic ad ap ta tion of ce real patho -gens to the mono cot y le don ous pri mary wall. Physiol. Mo lec u lar. Plant Pathol. 32: 33-47
Dori S., Solel Z., Barash I. 1995 Cell wall-de grad ing en zymes pro duced by Gaeumannomyces graminis var. tritici in vi tro and in vivo. Physiol. Mol. Plant Pathol. 46: 189-198
Enkerli J., Fe lix G., Boller T. 1999 The en zy matic ac tiv ity of fun gal xylanase is not nec es sary for its elicitor ac tiv ity. Plant Physiol. 121: 391-397
EsquerréTugayé M.T., Mazau D., Rumeau D. 1989 Sig nals and cell wall events in plantpatho gen in ter ac -tions. In NATO ASI se ries H35: 157-163
Furman-Matarasso N., Co hen E., Du Q., Chejanovsky N., Hanania U., Avni A. 1999 A point mu ta tion in the eth yl ene-in duc ing xylanase elicitor in hib its the â-1-4-endoxylanase ac tiv ity but not the elic i ta tion ac tiv ity. Plant Physiol. 121: 345-351
Giesbert S., Lepping HB., Tenberge KB., Tudzynski P. 1998 The xylanolytic sys tem of Claviceps purpurea: cy to log i cal ev i dence for se cre tion of xylanases in in fected rye tis sue and mo lec u lar char ac -ter iza tion of two xylanase genes. Phytopathology 88: 1020-1030
Großwindhager C., Sachslehner A., Nidetzky B., Haltrich D. 1999 Endo-1,4-D-mannanase is ef fi ciently pro duced by Sclerotium. Athelia rolfsii un der derepressed conditons. J. Biotechnol. 67: 189-203 Henrissat B., Bairoch A. 1993 New fam i lies in the clas si fi ca tion of glycosyl hy dro las es based on amino acid
se quence sim i lar i ties. Biochem. J. 293: 781-788.
Johansson M. 1988 Pec tic en zyme ac tiv ity of spruce. S and pine. P strains of Heterobasidion annosum. Fr. Bref. Physiol Mol Plant Pathol 33: 333-349
Kolattukudy P.E., Rog ers L.M., Li D., Hwang C.S., Flaishman M.A. 1995 Sur face sig nal ing in pathogenesis. Proc Natl Acad Sci USA 92: 4080-4087.
Krupinsky J.M. 1997a Ag gres sive ness of Stagonospora nodorum iso lates ob tained from wheat in the north -ern great plains. Plant Dis 81: 1027-1031
Krupinsky J.M. 1997b Ag gres sive ness of Stagonospora nodorum iso lates from pe ren nial grasses on wheat. Plant Dis. 81: 1032-1036
Lalaoui F., Halama P., Dumortier V., Paul B. 2000 Cell wallde grad ing en zymes pro duced in vi tro by iso -lates of Phaeosphaeria nodorum iso -lates dif fer ing in ag gres sive ness. Plant Pa thol ogy 49: 727-733 Lehtinen U. 1993 Plant cell wall de grad ing en zymes of Septoria nodorum. Physiol. Mol. Plant Pathol. 43:
121-134
Lotan T., Fluhr R. 1990 Xylanase., a novel elicitor of pathogenesis-re lated pro teins in to bacco., uses a non-eth yl ene path way for in duc tion. Plant Physiol. 93: 811-817
Lynch J.M., Slat er J.H., Bennett H.A., Harper S.H.T. 1981 Cellulase ac tiv i ties of some aer o bic mi cro or gan -isms iso lated from soil. J. Gen. Microbiol. 127: 231-236
Mac Ken zie C.R., Wil liams R.E. 1984 De tec tion of cellulase and xylanase ac tiv ity in isoelectric-fo cused gels us ing agar sub strate gels sup ported on plas tic film. Can. J. Microbiol. 30: 1522-1525
Magro P. 1984 Pro duc tion of polysaccharidede grad ing en zymes by Septoria nodorum in cul ture and dur -ing pathogenesis. Plant Sci. Lett. 37: 63-68
Mbwaga A.M., Menke G., Grossmann F. 1997 In ves ti ga tions on the ac tiv ity of cell wall-de grad ing en zymes in young wheat plants af ter in fec tion with Pseudocercosporella herpotrichoides. From Deighton. J. Phytopathol. 145: 123-130
Mendgen K., Hahn M., Deising H. 1996 Morphogenesis and mech a nisms of pen e tra tion by plant patho -genic fungi. Ann. Rev. Phytopathol. 34: 367-386
Miller G.L. 1959 Use of dinitrosalicylic acid re agent for de ter mi na tion of re duc ing sugar. Anal. Chem. 31: 426-428
Netzr D., Kritzman G., Chet I. 1979 b-(1,3 glucanase ac tiv ity and quan tity of fun gus in re la tion to Fusarium wilt in re sis tant and sus cep ti ble near-isogenic lines of musk melon. Physiol. Plant Pathol. 14: 47-55 Ol i ver R., Osbourn A. 1995 Mo lec u lar dis sec tion of fun gal phytopathogenicity. Mi cro bi ol ogy 141: 1-9 Riou C., Freyssinet G., Fevre M. 1991 Pro duc tion of cell wall-de grad ing en zymes by the phytopathogenic
fun gus Sclerotinia sclerotiorum. Appl. En vi ron. Microbiol. 57: 1478-1484
Robson G.D., Kuhn P.J., Trinci A.P.J. 1989 Ef fect of validamycin A on the roduction of cellulase., xylanase and polygalacturonase by Rhizoctonia solani. J. Gen. Microbiol. 135: 2709-2715
Royer J.C., Novak J.S., Nakas J.P. 1992 Ap par ent cellulase ac tiv ity of pu ri fied xylanase is due to con tam i -na tion of as say sub strate with xylan. J. In dus trial Microb. 11: 59-61
Saari E.E., Prescott J.M. 1975 A scale for ap prais ing the fo liar in ten sity of wheat dis eases. Plant Dis. Reptr. 59: 377-380
Schmidt C.S., Wolf G.A. 1999 Cellulase in the host-par a site sys tem Phaseolus vulgaris. L.)-Uromyces appendiculatus [Pers.] Link. Eur. J. Plant Pathol. 105: 285-295
Seevers P.M., Daly J.M., Catedral F.F. 1971 The role of peroxidase isozymes in re sis tance to wheat stem rust dis ease. Plant Physiol. 48: 353-360
Shaner G., Buechley G. 1994 Field eval u a tion of re sis tance to Septoria leaf blotch. p.173-176 in Arseniuk E., Góral T., Czembor P. eds)., Hod. Roœl. Aklim. Nasien. 38 (3-4)
Sharon A., Fuchs Y., An der son J.D. 1993 The elic i ta tion of eth yl ene biosynthesis by a Trichoderma xylanase is not re lated to the cell wall deg ra da tion ac tiv ity of the en zyme. Plant Physiol. 102: 1325-1329
Southerton S.G., Osbourn A.E., Dow J.M., Daniels M.J. 1993 Two xylanases from Gaeumannomyces graminis with iden ti cal N-ter mi nal amino acid se quence. Physiol Mol Plant Pathol. 42: 97-107
Sposato P., Ahn J.H., Walton J.D. 1995 Char ac ter iza tion and dis rup tion of a gene in the maize patho gen Cochliobolus carbonum en cod ing a cellulase lack ing a cel lu lose bind ing do main and hinge re gion. MPMI 8: 602-609
Stevens R.B. 1974 My col ogy Guide book. Uni ver sity of Wash ing ton Press., Seatte., WA. 703 pp Tonukari N.J., Scott-Craig J.S., Walton J.D. 2000 The Cochliobolus carbonum SNF! gene is re quired for
cell wall-de grad ing en zyme ex pres sion and vir u lence on maize. Plant Cell 12: 237-247
Waksman G., Keon J.P.R. 1989 Cellulolytic and pectinolytic en zymes from plant patho genic and non-patho genic fungi. Found. Biotechnical and In dus trial Fer men ta tion Res. 6: 197-206
Walton J.D. 1994 De cons truct ing the cell wall. Plant Physiol. 104: 1113-1118
Xu H., Mendgen K. 1997 Tar geted cell wall deg ra da tion at the pen e tra tion site of cow pea rust basidiosporelings. MPMI 10: 87-94
