• ·": I

· Crucifer Genetics doop~ra~ve~:~::. .. ~. _ :', .. Dept. of P~t )t~~ology, 1630 Lin~~,i--lJDiv~i~ W~consm, ~~I 58:708

. '.! -.\·.;, ,, . >:_.,:-t.~!':->·'•·, . ·"· ·,-·:',,(~~~-- . -

The Crucifer Genetics Cooperative gratefully acknowledges the support provided in the preparation of this workbook by OW-Madison's College of Agricultural and Life Sciences, the Department of Plant Pathology, and the University-Industry Research Program. The special assistance of graphic artist Mary G. Jondrow, of UIR, is also appreciatively noted.

-Paul H. Williams

© 1985 by Crucifer Genetics Cooperative, Dept. of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin

-~ .... '

SectionD

Diseases

~ Pathogen sources

~ Resistance screens

CrGC Crucifer Genetics Cooperative Dept. of Plant Pathology, 1630 Linden Dr., University of Wisconsin, Madison, WI 53706

(608-262-6496)

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN OR., UNIVERSITY OF WISCONSIN, HAOISON, WISCONSIN 53706 (603-262-6496)

ON 06-17-37 WILPAU

CRUCIFER DISEASE NAMES (Brassica and Raphanus spp.)

!.Alternaria diseases, black spot, leaf, stem or pod spots

2.Anthracnose 3.Aster yellows 4.Autogenic necrosis 5.Bacterial leaf spot

6.Bacterial soft rot, Erwinia

7.Bacterial soft rot, Pseudomonas

B.Black leg and Phoma root rot

9.Black mold rot

lO.Black root ll.Black rot

12.Black speck 13.Bottom rot, damping-off,

headrot,seedling root rot, wirestem, and basal stem rot

14.Cercospora leaf spot 15.Clubroot 16.Crown gall

17.Damping-off, Fusarium lB.Damping-off, Pythium l9.Downy mildew, staghead 20.Gray mold

2l.Light leaf spot

Symbol a

AB

CH AY AN(G) PSM

EC

PMM

LM

RST

AR xcc

BS RS

CB PB AT

F PT pp BC

PYB

1

Pathogen or Cause

Alternaria spp. (A. brassicae (Berk.) Sacc., A. brassicicola (Schw.) Wiltsh., A. raphani Groves & Skolko)

Colletotrichum higginsianum Sacc. Mycoplasma-like organism (MLO) Genetic disorder Pseudomonas syringae pv. maculicola

(McCulloch) Young et al. Erwinia carotovora (Jones) Bergey

et al. Pseudomonas marginalia pv.

marginalia (Brown) Stevens Leptosphaeria maculans (Desm.) Ces.

& de Not. (anamorph: Phoma lingam (Tode: Fr.) Desm.)

Rhizopus stolonifer (Ehr. :Fr.) Vuill.

Aphanomyces raphani Kendrick Xanthomonas campestris pv.

campestris (Pammel) Dawson Physiological Rhizoctonia solani Kuhn (teleomorph:

Thanatephorus cucumeris (Frank) Donk)

Cercospora brassicicola Henn. Plasmodiophora brassiae Wor. Agrobacterium tumefaciens (Smith

& Townsend) Conn. Fusarium spp. Pythium spp. Peronospora parasitica Pers.: Fr. Botrytis cinerea Pers.: Fr. (teleo-

morph: Botryotinia fuckeliana (dBy.) Whetz.)

Pyrenopeziza brassicae Sut.& Rawl.

22.Nematodes: a.Awl b.Cyst

c.Pin d. Root-knot e.Root lesion

f.Sting 23.Powdery mildew 24.Ring spot

25.Root rot, Phymatotrichum

26.Root rot, Phytophthora 27.Scab

28.Sclerotinia stem rot, watery soft rot

29.Southern blight

30.Tipburn 3l.Verticillium wilt

32.Virus diseases: a.Cauliflower mosaic b.Radish mosaic c.Turnip mosaic d. Yellows

33.White rust, staghead

34.White leaf spot, gray stem

35.Xanthomonas leaf spot

36.Yellows

D(N) H(N)

PA(N) M(N) P(N)

B(N) EP MB

PO

PM STS

ss

SR

TB(P) VD,VA

CAM ( V) RM( V) TUM(V) BWY(V) AC

PC

XCA

FOC

Dolichodorus spp. Heterodera schactii Schmidt

Reterodera cruciferae Franklin Paratylenchus spp. Meloidogyne spp. Pratylenchus pratensis (de Man)

Filipjev Pratylenchus spp. Belonolaimus spp. Erysiphe polygoni DC. Mycosphaerella brassicola (Duby)

Lindau Phymatotrichum omnivorum (Shear)

Dug. Phytophthora megasperma Drechs. Streptomyces scabies (Thaxter)

Waksman & Henrici Streptomyces spp.

Sclerotinia sclerotiorum (Lib.) dBy.

Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough)

Calcium deficiency Verticillium dahliae Kleb.

V. albo-atrum Reinke & Berth.

Cauliflower mosaic virus Radish mosaic virus Turnip mosaic virus B~et western yellows virus Albugo candida (Gmelin) Kuntze

(Peronospora sp. commonly present in staghead phase)

Pseudocercosporella capsellae (Ell. & Ev.) Deighton

Kanthomonas campestris pv. armoraciae (McCulloch) Dye

Fusarium oxysporum Schlecht. spp.

apathogen symbol: (G)=genetic disorder; (P)=physiological disorder; (N)=nematode; (V)=virus.

2

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON, WISCONSIN 53706 (608-262-6496)

r DO 05-08-85 WILPAU OCCURRENCE OF CRUCIFER DISEASES

Disease Pathogen Species Stage of crop Occurrenced Pathogen name symbole attackeda attackedb variabilityc

Alternaria diseases AB all V F 0 L black spot, leaf, stem or pod spots

Anthracnose CH a,ab,ac,r V F 0 H Aster yellows AY a,c V F R H Autogenic necrosis AN(G) a,b,c,r S V F R Bacterial soft rot EC all y c w H Bacterial soft rot PHH all y c w H Black leg LH all S Y F 0 w H Black mold rot RST a,c v 0 H Black root AR all S Y F 0 u Black rot xcc all S Y F c H Black speck BS a,c y 0 L Bottom rot, damping RS all S Y F 0 w u

off, headrot, seed-ling root rot, wire-stem and basal stem rot

Cercospora leaf spot CB a,ab V F 0 u ~ Clubroot PB all S Y F c H

Crown gall AT a,c y R u Damping off, Fusarium · F all s 0 Damping off, Pythium PT a,c s y 0 H Downy aildew, pp all S Y F c w u

staghead Gray mold BC a,c y F 0 u Light leaf spot PYB c,ac y F 0 u Ne1atodes:

Awl D(N) Cyst H(N) a,c s y R H Pin PA(N) Root-knot H(N) all s v R H Root lesion P(N) Sting B(N)

Powdery aildew EP all y F c H Ring spot HB a,c,ac y F c u Root rot, Phymato- PO a,c y F 0 u

trichum Root rot, Phytoph- PH a,c y 0 u

thora Sftb STS a,c v 0 H Sclerotinia stem rot, ss all V F 0 w u

watery soft rot Southern blight SR all y 0 u Tipburn TB(P) a,c y 0 H Verticillium wilt VD,YA a,c v 0 u

1

Virus diseases: Cauliflower mosaic CAH(V) all S V F 0 Radish 1osaic RH(V) r,a,c V F 0 Turnip mosaic TUH(V) all S V F c w Yellows BWY(V)

White rust, staghead AC all S V F 0 White leaf spot, PC a v 0

gray stem Xanthomonas leaf spot XCA all S V F c Yellows FOC all S V F c

aspecies attacked: a=.Brassica campestris, b=B. nigra, c=B. oleracea, ab=B. juncea, ac=B. napus, bc=B. carinata, r=Raphanus sativus.

u u u

H u

H H

bstage of crop attacked: S=seedling; V=vegetative growth to head; F=flowering and fruiting. cpathogenic variability: L=low; H=moderate; H=high; U=existence of variation is unreported. doccurrence: R=rare; O=occasional; C=common; W=widespread (Source Annon 1960; Walker 1952). •pathogen symbol (see DN 05-0B-85 WILPAU): (G)=genetic disorder; (P):physiological disorder; (N)=ne•atode; (V)=virus.

? ·-

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON, WISCONSIN 53706 (608-262-8638)

OSE 12-27-86 WILPAU

TIME COURSE AND ENVIRONMENTAL REGIMES FOR MULTIPLE DISEASE RESISTANCE SCREENING OF CRUCIFER SEEDLINGS

DAYS 0 7 14 21 fSEED fTRANSPI.ANT I 24"C I

28 I

35

X~~~~~ ~ ];:;:;:;:;:;:;:;:;:;:;:;:l;l:;:;:;:;tf

ruoS:~~RUM J--ir::.:{:::o:o:ol:l:l'"':l:l:='='=l:l:l'":l:l:o:o::;:;::o:o:ol:l:l'"':l:l:ml:l:l~f;..__-----,

LEPTOSPHAERIA MACULANS

RHIZOCTONIA SOLAN I

ERWINIA CAROTOVORA

PLASMODIOPHORA BRASSICAE

TuMV

PERONOSPORA PARASITICA

...

24"c 24"c

•o-

1-ii:::l:::l:::l::f 16"C 16"C

INOCI.A.ATION '?EAo INTERACTION PHENOTYPE

GREENHOUSE OR GROWTH

ROOM

~ 100" RH INCUBATOR

r

. 4'1'

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN 53706 (608-262-6496)

TARGET TISSUE LOCATIONS FOR MULTIPLE PATHOGEN INOCULATION OF CRUCIFER SEEDLINGS

xcc

AB = Alternaria AC = Albugo candida AR = Aphanomyces raphani EC = Erwinia carotovora

FOC = Fusarium oxysporum LM = Leptosphaeria maculans PB = Plasmodiophora brassicae PP = Peronospora parasitica

TUM = Turnip Mosaic Virus xcc = Xanthomonas campestris pv • campestris

DSTT 06-17-87 WILPAU

r CRUCIFER GENETICS COOPERATIVE

DEPT .. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN 53706 (608-262-6496)

DFOCS 05-07-85 BOSPAU

A NEW PATBOTYPE Of THE CRUCIFER YELLOWS ORGANISM CAPABLE Of CAUSING DISEASE IN COOL SOILS

Paul W. Bosland and Paul B. Williams

Pathotypes of Fusarium oxysporum f. sp. conglutinans cause yellows and wilt disease of various crucifers; race 1 is found predominately on Brassica, race 2 on Raphanus and races 3 and 4 on garden stock, Matthiola incana. Recently, a pathotype 4esignated race 5 has been found to be pathogenic against the monogenic dominant "type A" resistance gene introduced by Walker over 50 years ago (2). Race 5 appears to be restricted to two localities on a coastal plain of California. Unlike the common race 1 which causes yellows at soil temperatures normally above l8°C, race 5 is able to cause disease at soil temperatures as low as l2°C. This low temperature pathotype may be of concern to crucifer breeders in countries whose soil temperatures are normally too low to favor the expression of yellows incited by race 1. A survey of cabbage, broccoli, and radish cultivars from Japan, China, Northern Europe, North America, and Brazil for sources of resistance to race 5 is currently underway. Among 41 open pollinated and Fl cultivars of cabbage, as many as 75% of the individuals in a given cultivar were resistant to race 5, however some cultivars were completely susceptible. Radish cultivars also segregated for resistance. The cultivars Awa Long White, King Summer, Minowase Summer Cross No. 2, and Red Prince were resistant. There appears to be resistance in cultivars adapted to each country.

References

1. Armstrong, G. M. and Armstrong, J. K. 1966. Races of Fusarius oxysporus f. sp. conglutinans, race 4, new race and new host for race 1, Lychnis calcedonica. Phytopathology 56:525-530.

2. Ramirez-Villupodua, J., Endo, R. M., Bosland, P. W., and Williams, P. H. 1985. A New Race of Fusarius oxysporum f. sp. conglutinans that attacks cabbage with type A resistance. Plant Disease. (In Press)

1

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN 53706 (608-262-6496)

DACPR 05-13-85 EDWHAR

SELECTION FOR QUANTITATIVELY INHERITED RESISTANCE TO ALBUGO CANDIDA RACE 2 IN BRASSICA CAJIIPESTRIS, CrGC-1

Marlin Edwards and Paul H. Williams

Previous research with B. campestris has described a race­specific major gene Acl for resistance (James and Williams, 1980) or low reaction type on cotyledons to white rust, Albugo candida race 2 the common pathotype on Brassica juncea (Pound and Williams, 1963). Evaluation of heterogeneous populations of Brassica campestris reveals considerable variability in degree of reaction among susceptible genotypes when inoculated with A. candida race 2 under controlled conditions. This investigation was undertaken to determine if observed variability among "susceptible" individuals is under genetic control and to establish the degree to which selection for maximum quantitative resistance could reduce the response to infection.

The rapid-cycling experimental population of B. campestris, CrGC-1, was chosen to minimize the time required for this genetic investigation. The procedure involved isolating a number of genetically variable individuals which lacked the Acl gene for resistance and using them as a base population upon which selection pressure was exerted for low reaction to A. candida. Individuals to constitute the base population were screened for interaction phenotype (IP) to Albugo on a scale of 0 to 9 (where 0 = immune and 9 = very susceptible) (see CrGC-ID # DSAC--WILPAU) and self-pollinated to produce S1 progeny which were also evaluated to confirm that they possessed no major genes for immunity. Thirty-six individuals, exhibiting highly­susceptible interactions when inoculated with Albugo were selected upon this basis. These individuals were mass-pollinated to produce progeny for selection.

Selection was exercised·in two ways for three succeeding generations. The first was simply mass selection of individuals with the lowest reaction to Albugo. Since individuals were assigned discrete values for reaction based upon their phenotype, there were often more individuals in the lowest class than required to formulate the 36 - individual base for the succeeding generation. In this case, individuals were computer selected at random among those in the lowest class. The second selection procedure involved selection between and within maternal half­sib families. Progeny from each of the 36 - individuals in the preceding generation were grown in replicated blocks for evaluation of response to inoculation with Albugo. Selection was then exercised between families to choose the six families with the lowest reaction upon inoculation. Half-sib family selection was conducted to minimize bias due to non-genetic variation. For traits with high heritability, mass selection would allow greater selection pressure and would be expected to achieve greater gains.

1

After three cycles of selection, all populations were increased as two samples of 100 individuals which were intermated within samples to produce seed for evaluation. Evaluation was ~ conducted with inoculators and evaluators as factors in a randomized· complete block design. Analysis of variance revealed that differences between inoculators and evaluators were non­signific~nt. Mean values for populations are presented in Table 1.

TABLE 1. POPULATION MEANS FOR INTERACTION PHENOTYPE (IP) OF BRASSICA CAMPESTRIS, CrGC-1 AND ALBUGO CANDIDA RACE 2

POPULATION MEANS Cycle Method IP

Cycle 0 5.3 A Cycle 1 Half-sib Sel. 3.8 B Cycle 1 Mass Sel. 3.5 BC Cycle 2 Half-sib Sel. 2.9 BCD Cycle 2 Mass Sel. 2.3 CD Cycle 3 Half-sib Sel. 2. 1 . D Cycle 3 Mass Sel. 1.8 D

Mean separation based upon Duncan's Multiple Range Test, p =.05 IP: 1 = very low reaction type (high resistance); 5 = intermediate susceptibility; 9 = very highly susceptible

Duncan's Multiple Range Test for equivalence of mean values was ~ conducted and is also presented in Table 1. Response to selection is presented graphically below.

RESPONSE TO SELECTION s.o

LLJ a.. >-1- s.o 0 z LLJ ~ 4.0

KSF Selection: Gttn•-1.06 t.P./Cycle II ,., ltess Selection: G•tn•-1.16 l.P./Cycle '-,, 6 ,_ ..,,, .,,,; . ..,

,., •C't ftcotto, 'Gilt

0..

z 0 3.0 -1-u a: 2.0 a:: LLJ 1-z 1 • 0 - 3.0

CYCLE OF SELECTION

2

Average gain per cycle of selection was estimated as 1.16 IP/cycle for mass selection and 1.06 IP/cycle for half-sib family selection, based upon a simple linear regression model. Deviations from regression were non-significant.

These data illustrate that variability for reaction to A. candida race 2 among "susceptible" B. campestris, CrGC-1 individuals is due to quantitative genetic regulation. Furthermore, considerable gain in resistance may be achieved quite rapidly via mass selection when starting with a susceptible base population.

Seeds from each cycle of mass selection are available as CrGC stocks as follows: cycle 0, CrGC-62; cycle 1, CrGC-16; cycle 2, CrGC-17; cycle 3, CrGC-18.

Literature cited

Pound, G.S. and Williams, P.H. 1963. Biological races of Albugo candida. Phytopathology 53:1146-1149.

James, R.V. and linkage 70:776-799.

and Williams, P.H. 1980. Clubroot resistance in Brassica campestris. Phytopathology

Edwards, M. and Williams, P.H. 1985. Selection for 'minor' gene resistance to Albugo candida race 2 in a rapid­cycling population of Brassica campestris. Phytopathology (submitted for publication).

3

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON, WISCONSIN 53706 (608-262-8638)

DSTUHV 02-23-87 WILPAU

TURNIP MOSIAC VIRUS (TuMV)

HOSTS Cruciferae including Brassica sp., radish ornamentals, Nicotiana tabaccum, N. glutinosa, Chenopodium quinoa, spinach, pea and others. World-wide distribution on 20 dicot families.

DISEASE (Tomlinson, 1970) Mottling, black necrotic spots and ring spots on cc, mosaic with leaf distortion in aa, aabb & aacc; aphid transmitted.

DISEASE CYCLE

seed crop infected by aphids

HOST PROPAGATION .

1o inoculum; infected reservoir, ~ plants with aphids, weeds

mature crop infected by aphids

seedlings infected

by aphid feeding

Seed sown 0.5 em deep singly in separate pots of 12-pack multipots in 'peat-lite'; 24C; 18-24 hr, 250 ~moles-1m-2, irrigate daily with 50% Hoaglands sol'n to run off; grow plants for approx. 12-14 days or until 2nd true leaf has expanded.

1

TARGET TISSUE 1st, 2nd, 3rd, 4th true leaves just after they have expanded.

1st inoculation 2nd inoculation

INOCULATION Wash hands thoroughly with soap and water. Lightly dust 600 mesh carborundum on target tissues; inoculate by dipping the fore finger in the inoculum and gently rubbing the inoculum evenly over the leaf surfaces of the 1st and 2nd true leaves. For plants not showing symptoms after 10 days, inoculation is repeated on the 3rd and 4th true leaves.

INCUBATION Infectional environment; cover inoculated plants with a single sheet of moist newspaper for 10-20 hr; keep in host propagational environment.

Incubational environment; remove newspaper, maintain plants in host propagational environment 10 days, then observe for symptoms; those not showing symptoms are reinoculated, then all plants are evaluated 10 days later.

2

r \

~

EVALUATION Observe interaction phenotype (IP) on 20-day post-inoculation (PI) plants on both inoculated & non-inoculated leaves. Interaction phenotype; symptoms range from restricted, necrotic or chlorotic lesions on inoculated leaves to mottling and interveinal chlorosis of new growth to severe mottling, stunting and death. Scale: scored 0-9 at 20 days PI:

U-< ~ -J

C) ~ ~ < -J => ~ 0 z

O=absence of symptoms !=chlorotic or necrotic lesions on inoc. leaves 3=same as IP 1 + slight mottle and interveinal

chlorosis on > 20% of new growth, no stunting.

5=same as IP 1 + mottle on new growth, slight stunting but no leaf deformation

7=same as IP 1 + heavy mottling, deformation & stunting

9=same as IP 1 + severe mottle, stunting, death of plant

0

. ·. :. = . . •.:

3

CHLOROTIC OR NECROTIC LESIONS

3

5 7 9

~ .

MOTTLING

Disease index: 9

DI =L_ (i)(.i) n

i=O

n=total plants, i=IP class, j = no. of plants/class

Selection: susceptible plants, IP = 7-9; partially resistant, IP = 3-5; resistant plants IP = 0-1

HOST RECOVERY Selected plants may be vernalized directly in 12-pack pots or transplanted to larger pots or to the field; care must be taken to control aphids in these plants as resistant or partially resistant or tolerant phenotypes may be symptomless carriers of TuMV.

RESISTANCE A range of host-specific variation, strains exist in TuMV; resistance may be strain-specific.

1. B. oleraces, cc; most cvs. are susceptible; partial resistance, 'tolerance', exists in Badger Inbreds 1-13 (Pound et al., 1965), Russian cv. 'Burjucerutskoya-138, has monogenic dominant resistance to 2 strains but not all TuMV (Provvidenti, 1976); sources CrGC.

2. B. nspus, aacc, some European & N.Z. cvs. are resistant (Provvidenti, 1980).

3. B. campestris, aa, most ssp. & cvs. are susceptible; best sources are cvs. of aa.p from China (Provvidenti; 1980); multi-strain resistant cvs. from China are P.I. 391560, P.I. 418957, P.I. 418959, P.I. 419069; resistance is oligogenic or monogenic where a number of independently sorting factors appear to exist; P.I. 418957 & P.I. 419069 are resistant or partially resistant to TuMV strains C-1, C-2, C-3 & C-4; resistance appears to be strain-specific.

Sources: USDA, CrGC.

MULTIPLE PATHOGEN INOCULATION Plants may be inoculated on the roots with Foe, Ar or Pb, transplanted to 12-packs, inoculated on the cotyledons for Ac, Pp, Lm or Abr and multi-resistant survivors inoculated on the expanding true leaves with TuMV; later, plants can be incoluated with Kc and/or Ec. Interactions between TuMV infected plants and other pathogens have not been studied.

4

~-

VIRUS (Tomlinson, 1970) Potyvirus group. Flexuous, filamentous, 680-754 nm, sap transmissible to many species; aphid transmitted, non-persistent; world-wide distribution. ·

DIAGNOSTIC SPECIES Nicotiana tabaccum cv. 'White Barley': chlorotic local spots, 5-6 days, up to 5 mm, centrally necrotic, brown rim with chlorotic halo, not systemic. Brassica campestris, aa· r, turnip: chlorotic to necrotic local lesions, systemic vein clearing & flecking, mosaic, distortion, stunting. Chenopodiu• quinoa: chlorotic & necrotic lesions, 5-10 days after inoculation, systemic veinal flecks & spots. Others: cc.c, cc.b, Natthiola incana, (Flower breaking), Cheiranthus cheiri, Chenopodium a.DJaranticolor.

VECTORS 40-50 species of aphids, notably Nyzus persicae & Brevicoryne brassicae.

VARIABLIITY Strains: numerous host-specific strains exist; variation in virulence has been reported (Provvidenti; 1980). Serology: weakly to moderately immunogenic; antisera with titer l/512 from cc.c in precipitin tube tests, 0-85% NaCl diluent (Shepherd & Pound, 1960).

STABILITY In vitro stability differs with isolates. Thermal inactivation point 62 C. Dilution end point in sap 10-3-10-4. Infectivity retained at 20 C, 3-4 days. Infective sap kept at 2 C for several months.

INOCULUM Initial: dried virus in leaves is ground into buffer. Propagation, B. campestris, turnip cv. 'Presto' (S); other species; N. glutinosa, aa.pe 'Tendergreen'; aa.p cvs. Produce 12-14 day-old-plants of 'Presto' turnip as described under host propagation; inoculate plants and maintain them in an aphid-proof screened isolation cage under environment for host propagation

5

For.mulation: from heavily infected plants, remove portion of mottled and distorted leaves; using a measuring grid to estimate infected leaf area, add 4 cm2 leaf tissue per 5 ml 0.05M phosphate (K+) buffer, pH 7.0, to a mortar, grind tissue with pestle; freshly ground leaf sap is the inoculum.

STORAGE Remove heavily infected leaf tissue, chop into 0.2-0.5 em wide strips on clean paper using razor blade, place in a drying pan in desiccator with CaCl2 or silica gel drying compound. Dry tissue for several days, then transfer dry pieces to screw cap vials containing drying compound in the bottom. Store dried virus & vials at 4C. Stored virus will keep for years. Small amounts of virus & leaf can be dried and stored in #00 gelatin capsules.

SOURCES ATCC, PHW, RP (various strains).

QUANTIFICATION Infectivity titration using local lesion assay host can be run, but is not necessary for routine screens. Collect & grind 1 gram of infected leaf tissue in 10 ml buffer; prepare 6 serial, 10-fold dilutions of the virus extract. Inoculate each dilution to randomized half leaves of an "assay species": H. tabaccu.lll, C. quinoa or C. B.lllarsnticolor, replicate each dilution on 5 half-leaves; inoculate; incubate for 5-10 days; count local lesions on each half leaf & plot infectivity vs. dilution; a suitable dilution of infected leaf tissue can then be selected.

6

(· CRUCIFER GENETICS COOPERATIVE

DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON, WISCONSIN 53706 (608-262-6496)

BLACI ROT

Xanth~MD~Jas ca~tris pv. ca~tris {Pal.) DOM. {XCC)

HOSTS Cruciferae including all cultivated and many wild species (Hayward and Waterston, 1965).

DISEASE Seed-borne, splashing water and wind spread. A vascular bacteriosis causing yellowing of leaves and brown to black discoloration of the veins. Invades roots, ste1s, leaves and flowering parts. Leaf lesions, predominantly V-shaped marginal lesions expanding down veins from hydathodes, eventually beco•ing systemic. Lesion tissue desiccates and turns brown. Succulent infected tissues frequently becoae invaded by soft rot bacteria, Erwinia carotovora (EC).

DISEASE CYCLE 1° Inoculu; Infested seed,

Seedcrop

~ infected.weeds, ~ / crop restdue.

syste1ic invasion Seedling often undetected. infection t via l"d coat.

Spread in crop f via rain splash Spread in seed (possible aerosols), bed, rain splash. insects, 111achinery, / irrigation water.

HOST PROPAGATION

'-. Transplants, "-----transplanting

injury.

Seedling Screen: Seed sown 0.5 em deep, singly in pots of 12-pack aultipot in peat-lite; 24 c, 18-24 hr, 250~mol s· 1m- 2 ; for S-7 days or until cotyledons fully expanded. Irrigate with 0.5 x Hoaglands daily to run off.

Field Screen: Uses aature plants. Grow seedlings in suitable seed beds or in transplant trays. Transplant to field SO cs apart in rows. Grow for 4-6 weeks, use normal

DSXCC 05-13-85 WILPAU

fertility and recom1ended pest controls.

TARGET TISSUE Seedling Screen: Cotyledons, 24-28 hr after expansion. Field Screen: Leaves of 4-6 week field grown plants.

INOCULATION Seedling Screen: Briefly dip round wood tooth picks in inoculum, then make a single

puncture through the 1idvein of the cotyledon near margin.

~ .... ..,.,.~, ~-Jr•"'r''"

Field Screen: Using an ultra low voluae pesticide applicator (Perry and Williaas, 1984), 'HINI ULYA', held approxi1ately 10-25 ca above plants and adjusted to deliver 30-40u droplets of inoculum at a flow rate of 30 al/min, advance over plants in row at 1 H/sec. This will deliver approximately 0.25 ml inoculum per plant at the SO em spacing in the row.

Time of application must be very early morning when heavy guttation and dew have formed on plants. Application should be done in still air if possible. Bacteria in microdrops enter guttation water and are drawn into plants as transpiration occurs during day. Care should be taken not to dislodge droplets at leaf aargins.

Alternate Inoculators: Hand-held pressure tank sprayers with fine spray nozzels or power-driven air blast back-pack pesticide sprayers are as effective as the ULVA. With these, higher volumes of dilute inoculum are used, see Inoculum Formulation section.

"··* ,_,. .,...., ...

INCUBATION Seedling Screen: Continue to grow plants in 12-packs in same environ1ent as pre­

inoculation. Grow plants 14 days. Field Screen: Continue to grow plants using normal practices, pay particular attention

to ainimizing insect injury.

EVALUATION Seedling Screen: observe interaction phenotype (IP) at 7 through 14 days post inoculation

2

(,....._

(PI). On cotyledons; IP will range from no visible signs or symptoas apart from injury hole, to progressive chlorotic and necrotic tissue collapse, systeaic invasion and death of seedling. Scale: Plants scored 0-9 at 7-14 days. IP scores on susceptible individuals will

progress eore rapidly than on resistant. 0 = No sy1ptoms around point of injury and inoculation. 1 : Harrow area of restricted necrosis around inoculation point, no chlorosis. 3 = Limited necrotic tissue collapse, surrounding inoculation point. Very

restricted marginal chlorosis or none, no stunting. 5 = Limited necrotic tissue collapse extending toward petiole, restricted •arginal

chlorosis with li1ited vein blackening in lesion. 7 = Necrotic and chlorotic lesion collapse extending toward, petiole, darkened

veins in lesion, stunting of new growth. 9 = Extensive chlorosis and necrosis spreading frot inoculation point into petiole,

vein darkening into petiole, systeaic invasion, stunting, death of seedling.

3 .5 1 9

···&fa~·· ~ .......... : :·,·:;. . ~·,

Field Screen: Observe IP at 14-35 days PI on all inoculated leaves and on new leaves of plant, IP will range fro• no sy1ptoas through progressive marginal necrosis and chlorosis to severe syste1ic invasion followed by soft rot. Scale: Plants scored 0-9, 21-28 days PI.

0 : No sy1pto1s, noraal growth. 1 : Hinute necrotic lesions at hydathodes to discrete vein-restricted necrotic

aarginal panels with dark edges. Panels 0.5 to several ca long x O.S-2 ca wide, no chlorosis beyond aargin of necrotic lesion.

3 : Small marginal Y-shaped lesions, O.S-2 ca with narrow diffuse chlorotic margins, sometimes necrotic panels with diffuse chlorotic margins.

5 : Stall to aediua V-shaped marginal lesions, necrotic centers, diffuse chlorotic margins, internal and marginal veins blackened. Lesions rarely progressing half way to aid-rib.

7 = Progressi~g marginal · lesions, frequently reaching mid-rib, prominant vein blackening.

9 = Rapidly progressing marginal lesions frequently coalescing to give scorched leaf, systemic invasion of plant, frequently accoapanied by soft rot, severe· stunting, death of plant.

0 3 s 7 9

Note: Insect control is very important because when XCC is introduced directly into veins of resistant plants, IP=0-1, partially resistant phenotypes, IP=3-S, will be

~ expressed (Staub and Williams, 1972).

3

9 ~ (ixj)

Disease Index: DI= ~i=.._O __ n

n=total plants, i=IP class, j:number plants/class. Sel~ction: Susceptible plants, IP=7-9; partial resistance, IP=3-5; resistant, IP=0-1. Controls: Susceptibles every 2 rows, sxxsxxs.

HOST RECOVERY Seedling Screen: Remove inoculated cotyledons, susceptible plants rarely survive.

Growing plants at 10-18 C may permit plants to outgrow XCC. Partially resistant and resistant plants may be grown on. Care must be taken to minimize spread of XCC from inoculated plants to others, bacteria will persist in partially resistant plants.

Field Screen: Susceptible plants rarely survive, soft rot will invade lesions. Harvest selected plants by removing head, let stump callus over for 5-10 days, pot up stump and encourage axillary shoot growth. Treat cut surfaces of stump and soft rot with dusting of streptomycin. Hot water treat seed, at 50 C for 25-30 min, from XCC inoculated plants.

RESISTANCE Host cultivated crucifers are susceptible to XCC. B. oleracea, cc, is especially susceptible, oligogenic recessive resistance with modifiers in cv. 'Early Fuji' (Takii and Co., Ltd.) (Williams et. al., 1972), Early Fuji resistance transferred to Badger Inbreds 14-20 (CrGC). cv. 'Hancock' (Alf Christianson Seed Co., Northrup King Seed Co.) is resistant (IP=0-1). 'Fuji' resistance is expressed against common XCC isolates in USA, Japan, Australia, India and Brazil. Brazilian cv. 'Locou' is reported resistant. Polygenic resistance in cauliflower (Sharma, et. al., 1972).

MULTIPLE PATHOGEN INOCULATION Inoculation of seedlings with XCC and other pathogens is unexplored, needs further study. Following inoculation of seedlings with various pathogens, FOC, AR, PB on roots, AC, PP, LH, AB on cotyledons and TUM or EP, plants may be transplanted to field and inoculated with xcc.

4

BLACI ROT

Xantb,..as ca~tris pv. cupestris (Pu.) Dot. (XCC)

ORGAMISI Aerobic, graa negative rod, 0.7-3.0 x 0.4-0.5p, aotile by single polar flagellut. Vascular parasite confined primarily to xylet (Haywood and Waterston, 1965), widely distributed on crucifers.

liFE CYClE Reproduction by siaple fission.

APPEARANCE In culture: Colonies are yellow, convex, shiny and of a sliay, aucoid consistency. Microscopic: Rod 0.7-3.0 x 0.4-0.Su. Hotile by single polar flagellua.

VARIATION Stability: Hoderately variable in culture, little loss of virulence in culture. Mechanists: Transduction, specific phages are known (ATCC). Phenotypic: Strains with varying xanthan gut producing capacity are known (NRRC). Pathotypes: A few variants have been reported, XC var. artoracia on Radicula artoracia,

XC var. aberrans is 10re virulent on cc.b, leaf spotting and stomatal invading and low teaperature pathotypes have been reported.

INOCUI.UII Initial: Lyophyllized cultures or stored cultures. Propagation: Aseptically break lyophyll vial. Transfer pellet to PDB in 125 11 flask.

Shake 60-80 cycles/min. at 28 C for 24 hr. Subculture by streaking to PDA plates to verify culture hotogeneity. Incubate plates 48 hr at 23 C. Larger volu;es of inoculu• prepared by transferring 24 hr shake cultures or 48 hr plate colonies to BOO ntl PDB in 2300 Ill Fernbach flasks and shaking thea for 48 hr at 28 C, 60-80 cycles/ain.

Quantification: To quantify nutber of cells in 48 hr culture, use dilution plate and turbidotetric ;ethods. All glassware aust be sterile for these aethods. Dilute 48 hr shake culture with sterile distilled water (sdw) until suspension gives a 0.1 00 at

~ 600 na in a Baush and Loab Sectronic 20, spectrophotoaeter (this is approxiaately s-8xi07 cells/all. Prepare 5 10-fold serial dilutions of this 0.1 OD preparation in

5

tubes containing 9 ml of sdw. Transfer 0.1 ml of each dilution to each of 3 PDA plates. Spread saaples thoroughly over plate using bent glass rod. Incubate inverted plates at 28 c. Count colonies in the dilution range of 30-300 per plate. Estimate nuaber of viable cells in original 48 hr culture of OD=O.l dilution.

Fortulation: 1. Seedling Screen: 48 hr shake culture may be used directly to soak toothpicks in. 2. Field Screen: Field inoculation with the ULVA uses 48 hr shake culture of PDB

without dilution. 3. Alternate Inoculators: Dilute 48 hr shake cultures 1:20 in water to which 0.05 g

HgS04/liter has been added to stabilize the bacteria.

STORAGE Long Ter1 (years): Lyophilization, 48 hr PDA slant cultures grown at 28 C. Add 1 ml

sterile 20% skim milk, suspend cells thoroughly. Dispense 0.2 ml of suspension to freeze-dry vials. Freeze, vacuum dry, seal, label, store at 4 C or -20 C. Cells may also be stored on silica gel. Hake a sterile mentruum by adding 2 g dry milk to 20 ml water and autoclave. Autoclave 3 g of 40 mesh silica gel in vials and dry. Hake a cell suspension with the menstuum and add 0.5 ml to chilled vials of silica gel in an ice bath, thoroughly shake and store at 4 C.

,;~1: · c,/f'w~c.

s-f.,-,"1• ,; 2.0 ;c. ~,c,._ .~ .. ,

,.,·tic .

Short Ter• (tonths-years): On PDA under oil at 4 C.

SOURCES

-6&1.1", ..... ;0 .... •-' .. ;lie.

ATCC, ICPB, NRRC (industrial strains), L. L. Black, N. W. Schaad (large collection of isolates), J. 0. Strandberg, CrGC.

REFERENCES Alexander, H., P.H. Daggett, R. Gherna, S. Jong, F. Simone. 1980_ ATCC Hethods I.

Laboratory Hanual on Preservation Freezing and Freeze-Drying. ATCC. Rockville. 51 pp. Hayward, A. C. and J. H. Waterston. 1965. Xanthomonas campestris. CHI Descriptions of

Pathogenic Fungi and Bacteria. No. 47. Commonw. Hyco!. Inst., Kew. Perry, J. B. and P. H. Willia1s. 1984. Controlled droplet applicators for inoculation

6

·~ l

r with plant pathogens. Plant Disease. 68:107-108.

Sharma, G. R., Y. Swarp, and S. S. Chatterjee. 1972. Inheritance of resistance to black rot in cauliflower. Can. J. Genet. Cytol. 14:363-370.

Staub, T. and P. H. Williams. 1972. Factors influencing black rot lesion development in resistant and susceptible cabbage. Phytopathology. 62:722-728.

Tuite, J. 1969. Plant Pathological Hethods. Burgess, Minneapolis. 239 pp. Williams, P. H. 1980. Black rot: a continuing threat to world crucifers. Plant

Disease. 64:736-742. Williams, P. H., T. Staub and J. C. Sutton. 1972. Inheritance of resistance in cabbage

to black rot. Phytopathology. 62:247-252.

7

r

~ I

PURIFICATION (Tomlinson, 1964) Extract tissue at pH 7.5 in 0.5 M borate buffer containing 0.001 M NaEDTA + 1% thioglycolic acid; add n-butanol to 8.5%, centrifuge twice at low speed; sediment by high speed centrifugation; resuspend pellet, virus, in 0.05 M borate (pH 7.5); do all steps at 4C.

REFERENCES

Fjellstrom, R. J. and P. H. Williams. 1986. Genetics of turnip mosaic virus resistance in Brsssics csmpestris. (in preparation).

Pound, G.S., P.H. Williams, and J.C. Walker. and yellow resistant inbred cabbage varieties. Agr. Exp. Sta. Res. Bull. 259. 30p.

1965. Mosaic Wisconsin

Provvidenti, R. 1980. Lectures on resistance to viral diseases of vegetables. 118 pp. Dept. of Plant Pathol. NY State Agr. Exp. Sta., Cornell Univ., Geneva, NY 14456.

Shepherd, R.J. and G.S. Pound. 1960. Purification of Turnip Mosaic Virus. Phytopathology 50:797-803.

Tomlinson, J.A. 1964. Lettuce Mosaic Virus.

Purification and properties of Am. Appl. Biol. 53:95-102.

Tomlinson, J.A. 1970. Turnip Mosaic Virus CMI/A.A.B. Descriptions of Plant Viruses. No. 8. Commonw. Mycol. Inst., Kew.

7

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON, WISCONSIN 53706 (608-262-6496)

ClUB ROOT

PlaSIOdiQPhora brassicae Noronin (PB)

HOSTS Cruciferae, especially important on Brassica, common on weeds and Raphanus (Buczacki, 1979; Calhoun, 1958; Karling, 1968; Walker, 1963).

DISEASE Fusifort or spherical enlargetents and malformations (galls) on 1°, 2° and 3° roots, sometimes extending to the hypocotyls, rarely into the stet and leaves. Infected plants wilt in direct sunlight. Plants are more or less stunted and occasionally die from root gall invasion by secondary soft rot organists.

. . ). \

DISEASE CYCLE \ 1° Inoculut Cysts in soil.

/ 1° zoospores.

DSPB 05-13-85 WILPAU

""' Necrosis of galls by secondary organists.

r \

1° infection, root hair and epidermal cells

I,

Wilting, stunting disruption of vascular function.

\ J

2° zoospores.

/ Gall formation, 2° infection of cortical hyperplasia, ..___ and vascular tissue. hypertrophy.

HOST PROPAGATION Space seed 1-2 c• apart on moist filter paper, inclined to guide roots downward. Grow 3-5 days, 20-25 C, 13-24 hr, 250p;ol s· 1;· 2•

TARGET TISSUE Seedling roots. Remove seedlings from filter paper, permit adhering filter paper to stay

~ with seedling. I \

1

INOCULATION 1. Dip roots briefly in PB spore suspension and transplant singly into multipot 12-packs

in aoist, finely screened (2 11 aesh) 1:1 peat aoss:veraiculite, 'peat-lite'.

,.,. ~~- ,;., 12-l .... lc. ;,. pc•4-li1c.

2. Dust roots to thoroughly coat them with PB spore mix (see Inoculum Formulation-2.). Plant as above in peat-lite.

~~ 1-l . .. . .. -? ~ -'P .... • . . . --4> " ~ ,~, .... t-"?

,.~ ... r,;, st•'• ,..,;. - .. sa•-'''*'' ,..o-r.

INCUBATION Irrigate daily to runoff with water for 5 days then daily with 0.5 x Hoaglands solution. 20-25 C, 18-24 hr, 250ptol s· 1m· 2 • Grow plants 3-5 weeks.

EVALUATION Lift plants and shake excess soil into bucket. Wash roots thoroughly, place roots in shallow tray of water for viewing and scoring.

INTERACTION PHENOTYPE (IP) - Fibrous rooted forms Syaptots: Range fro• ainute to larger nodular galls (1-5 ma) on roots, to fusiform galls

of increasing size extending to 2° and 3° roots and hypocotyl. Depending on plant age at evaluation, aore or less stunting of tops aay occur.

Scale: Plants scored 0-9 at 3-5 weeks, based on root symptoms only. o : No syaptoas. 1 = Saall nodular galls. 3 : Larger nodular or spherical galls.

2

S-7 : Increasingly large fusifora galls on lateral and main root. 9 : Large galls extending into hypocotyl.

0

9 2: (ixj)

Disease Index (DI): DI= :..i=.::...O __ n

3

n=total plants, i=IP class, j:nuaber plants/class

5 7 9

Selection: Susceptible controls, IP=7-9; partial resistance, IP=3-5; resistant plants, IP=0-1, plants 1ay be recovered.

HOST RECOVERY Selected survivors eay be transplanted to pots and grown at 20-25 C. Take cuttings of partially resistant or susceptible plants. If planting to field, roots may be dipped in a suspension of 0.2 g/liter benoayl fungicide.

RESISTANCE A wide range of pathotypic variation exists in pathogen populations. Few sources of effective resistance in most crops. 1. B. oleracea: Host cvs. susceptible. Resistance in cc is generally oligogenic and

recessive. Sources: cc.c, 'Badger Shipper' (Harris-Horan Seed Co., Ferry-Harris Seed Co.); 'Oregon-100', '-123', '-140', '-142' (J. R.Bagget); cc .b, 'WAWI -1', '-2' (R.L.Gabrielson, CrGC); 'WIPbR-1', 'WIPbR-2' (CrGC).

2. B. ca1pestris, aa, and B. napus, aacc, resistance generally oligogenic and doainant. Pb1/Pb1, Pb2/Pb2, Pb3/Pb3 in aa stocks (CrGC) (James and Williams, 1980).

3. R. sativus, rr, nuaerous cvs. carry varying levels of resistance. Sources 'Read', 'Saxafire' (Vander Ploeg Seed Co.). Interspecific (cc x aacc, Chiang et. al., 1980; aacc x aa, Laaaerink, 1970) and intergeneric (rr x cc, HcNaughton and Ross, 1978) have been achieved.

Kultiple Pathogen Inoculation Plants inoculated with PB aay be inoculated on cotyledons with PP, AC, L~ or AB. PB resistant plants aay be inoculated later with TUH, XCC, EC and EP (Willia•s and Leung, 1981). Inoculation with PB and FQC is unexaained. Plants may be examined for TB in field.

3

CLUB ROOT

PlaSIOdiopbora brassicae Moron. (PB)

ORG.\111511 Obligately biotrophic endocellular parasite, Plasmodiophorales, Hyxoaycota. Soil-borne, root invading.

LIFE CYCLE (Ingral and Tommerup, 1972)

APPEARANCE

- ""-?.--0. ~-~ '-::: .... ,t-. ,t ,,__...~.,.~

®

.. ~·.:o·:·:~ :C?·:. ,j,,f.,swtoJri..-

Macroscopic: Organis1 is aicroscopic. In mass, the concentrated purified resting cysts (spores, sporangia) are grey to tan in color.

Kicroscopic: Sections of clubroot galls show vegetative plasmodia and aature resting cysts in typical "Krankheitsherd".

Plasmodia and secondary sporangia may be seen in root hairs and epidermal cells.

1° and. 2° zoospores are 2.5-3.Sp, anteriorily biflagellate.

4

r Cysts (resting spores, resting sporangia) are 3.2u, spherical, uninucleate, 1inutely spiny.

VARIATION Based on host range. Pathotypic variation very great. Virulence phenotypes of local populations largely dependent on local variation in host genotypes in crop and weed species. Races: Various scheaes exist; differential reactions on cc.c, 'Badger Shipper' and

'Jersey Queen' and on aacc.r 'Laurentian' and 'Wilhelasburger' define 16 races (Williaas, 1966). A European Clubroot Differential (ECD) set of 5 cvs. each of aa, aacc, and cc is widely used to differentiate virulence profiles of PB populations (Buczacki et. al., 1975). 'Differential' seed available fro• GRD.

INOCII.III Initial: Frozen or fresh clean clubroot galls fro• infections 1ore than 5 weeks old. Propagation: Produce clubroot galls under standard conditions described herein or

collect fresh 'aature' clubs fro• the field. Preparation: Triturate fresh or frozen clubs, LOO gin 400 al water, 2 tinutes at high

speed in blender. Filter through 4 layers of cheesecloth.

Retriturate and refilter pulp if desired. Centrifuge filtrate 2000 x g for 7 minutes.

Botto; layer in centrifuge tube is debris, white layer is host starch, top grey layer is cysts. Suspend cyst layer in water, recentrifuge. Repeat 2-3 tiaes if needed to clarify or concentrate cyst suspension. Exaaine cysts under 1000 x magnification.

Viable cysts have unifora fine granules. Inviable cysts have irregular granules. Germinated cysts are e~~pty and show exit pore.

Quantification: Quantify percent viable cysts using heaocytometer. Adjust suspension to

5

109-101° viable cysts/ml. Store in vial at 4 C.

ForiUlation: 1. Water suspension of cysts for root dip inoculation adjust to 108 viable cysts/mi.

After storage, for even a few days or weeks, reexaaine percent viable cysts.

~.

. ·'· - :,·_ . -£·~ ... . .. ,, ....

;.,.c.._/ .. ~ , ... ,. .. , ·-2. Cyst Hix: Alternate formulation for more stable storage and shipaent, aix 1:1

finely sieved, sterilized peat-aoss and black organic peat soil. Dry soil aix to approximately 0.36 g/cc.

Quantify viable PB cysts to 109 viable cysts/ml HzO and mix very 1 al PB cysts/10cc soil aix. Cyst mix will be slightly moist.

thoroughly

Store cyst mix in jar at 4 C. For mailing, seal cyst mix in an autoclavable heat sealable aoisture-proof plastic fila.

STORAGE Long Ter1 (years?): As frozen clubs, -20 Cor lower. Thawing and refreezing may result

in deterioration of cysts. Desiccation may be damaging to viability (more testing needed on alternate long term storage methods).

6

Short Ter1 (~eek to a fet years): Cyst mix at 4 C (requires further testing), concentrated aqueous cyst suspensions 109-1010/ml at 4 C. Storage life variable, depends on condition of cysts and buildup of putrifaction organisms in cyst suspension (more research needed).

SOURCES Populations of varying virulence and degrees of homogeneity can be obtained from members of the rnternational Clubroot Working Group, write GRD; CrGC (race 6).

TEST FOR P8 GER"INABILITY AND INFECTIVITY A rapid aethod of exaaining PB cyst preps for infectivity. Use seedling of 8. oleracea, cc.c, 'Jersey Queen' or other susceptible cv. or any other suitable crucifer (Williams et. al., 1971). Space seed 1-2 cm apart on moist filter paper inclined in inverted petri dishes to maintain a water reservoir. Germinate seed 2-3 days. 20-24 c, 13-24 hr, 1S0-2SO~mol s-t.-z.

Pipette Sul quantified cyst suspension to center of a 1 ca of Whatman 11 filter paper. Cysts ~ill form a pellet. Place cyst pellets in petri dish to keep them slightly aoist. Fill 1 draa 'Opticlear' vials with hole in top with Hacfarlane's solution. Place PB cyst pellet in vial and insert seedling root through hole in vial cap.

Place vials in clear plastic or glass boxes at 22-25 C, 24 hr, 50-150umol s- 1.-2 •

Examine whole roots under 100-250 x magnification for PB plasmodia and 2° sporangia in root hairs. Infection verifies infectivity of preparation. Examine scraping from cyst pellet for germinated cysts. Empty cysts verifies geminibility of preparation. After viewing, seedlings may be planted.

7

REFERENCES Buczacki, S. T., H. Toxopeus, P. Hattusch, T. D. Johnston, G. R. Dixon, and L. A.

Hobolth. 1975. Study of physiologic specialization in Plasmodiophora brassicae. Proposals for attempted rationalization through an international approach. Trans. Br. Hycol. Soc. 65:295-303.

Buczacki, S. T. 1979. Plasmodiophora brassicae. C.H.I. Descriptions of Pathogenic Fungi and Bacteria, No. 621. Commonwealth Mycological Inst., Kew.

Chiang, B. Y., H. S. Chiang, W. F. Grant and R. Crete. 1980. Transfer of resistance to race 2 of Plasmodiophora brassicae fro• 8rassica napus to cabbage (8. oleracea ssp. capitata) IV. A resistant 18-chromoso•e B1 plant and its B2 progeny. Euphytica 29:47-55.

Calhoun, J. 1958. Clubroot disease of crucifers caused by Plasmodiophora brassicae Woron. Phytopath. Paper No. 3. lOB pp. Com1. Hyco!. Inst., Kew.

Jues, R. Y. and P. H. Williams. 1980. Clubroot resistance and linkage in 8rassica campestris. Phytopathology. 70:776-779.

Karling, J. s. 1968. The Plasmodiophorales. 2nd ed. Hafner Pub. Co., New York. La111Berink, J. 1970. Interspecific transfer of clubroot resistance fro11 8rassica

campestris L. to 8. napus L. N. Z. J. Agr. Res. 13:105-110. McNaughton, I. H. and C. L. Ross. 1978. Interspecific and intergeneriG hybridization in

the 8rassicae with special emphasis on the i1prove1ent of forage crops. Scottish Plant Br. Stn. 57th Ann. Rept. pp. 75-110.

Walker, J. C. 1968. Plant pathology. 3rd ed. HcGraw-Hill, New York. 819 pp. Willia11s, P. H. 1966. A syste• for the determination of races of PlaslBodiophora brassicae that infect cabbage and rutabaga. Phytopathology. 56:624-26.

Williams, P. H. and H. Leung. 1931. Hethods of breeding for multiple disease resistant chinese cabbage. pp. 391-403. InN. s. Talekar and T. 0. Griggs (eds.). Chinese Cabbage, AVRDC, Shenhua, Taiwan.

Williams, P. H., S. J. Aist, and J. R. Aist. 1971. Response of cabbage root hairs to infection by Plasmodiophora brassicae. Can. J. Bot.

8

CRUCIFER GENETICS COOPERATIVE DEPT.OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON, WISCONSIN 53706 (608-262-6496)

DOWNY "ILDEII

Perooospora parasitica Pers. ex. Fr. (PP)

HOSTS Cruciferae (Yerkes and Shaw, 1959), numerous cultivated and wild species.

DISEASE Soil-borne (oospores), wind and rain spread (conidia). Fine, dense to sparsely packed white •at usually on chlorotic and partially necrotic irregular lesions on leaves. Sometimes hypertrophic lesions on stems and flowers, often found in sa111e gall tissue as AC. Fir• necrotic areas in storage parenchy1a, roots, heads, flower stalks.

DISEASE CYCLE 1° Inoculum

Oospore production in host tissue.

oospores in soil? ~ Syste•aticall y invaded plants.

Oospore ger•ination, infection of roots? t Sporangia! Intercellular

Production of ~ germination spread to stems sporangia, on leaves. and leaves. so1e invaded \ ) cells necrotic, t others not. Intercellular

~ colonization of leaf, ""--- root and stems,

haustoria in parenchyma.

HOST PROPAGATION Seed sown 0.5 em deep singly in individual pots of 12-pack multipots in 'peat-lite', 24 C, 18-24 hr, 250)1101 s·lm- 2 for 5 days or until cotyledons first expand. Irrigate with 0.5 Hoaglands daily to run off.

1:::! - p•ck

'"'"/f',uf~~~~~~

TARGET TISSUE r· Cotyledons, 24-48 hrs after expansion.

1

DSPP 05-13-85 WILPAU

INOCUI.ATIOII Place a 10ul drop of conidial suspension on each 1/2 cotyledon using aicropipette. Prevent droplets fro• evaporating. Gently agitate inoculu• to keep conidia from settling.

INCUBATION Infectional Environtent: Haintain inoculu1 droplets on cotyledons 12-18 hr, 16 C, 100%

RH. Place plants in dew chaaber in dark.

Incubation Environ~ent: Sale as host propagation environment. Reaove plants froa dew cha1ber and·grow thea for S days.

Interaction Phenotype (IP) Inducing Environ~ent: Return plants to dew chamber at 6 days post inoculation (PI) and 1aintain sa1e conditions as infectional environaent, 16 C, 100% RH, for 24 hr.

EVALUATION Observe IP on 7 day PI cotyledons. Interaction Phenotype: Rates for both sympto1s ranging fro• a 'hypersensitive' necrotic

flecking at the site of the inoculu• droplet to irregular narrow streaks of internal necrosis associated with 1ore or less chlorosis. Signs of the pathogen range from no sporulation to very sparse, through increasingly heavy sporulation on both the top and botto1 of the cotyledons. Host highly compatible IP's have little or no necrosis, none to light sporulation on the upper, adaxial, surface of cotyledon with heavy sporulation on the lower surface.

Scale: Plants scored 0-9 at 7 days PI after having been at 16 C, 100% RH, for 24 hr. 0 : No SYIPtOIS or signs of PP. 1 : Very minute to larger scattered necrotic flecks under inoculu1 drop, none to small

amounts of necrosis on lower cotyledon surface, no sporulation. 3 = Very sparse sporulation, 1 to few conidiophores on upper or lower surfaces.

Necrotic flecking often present. Tissue necrosis present. S = Sparse scattered sporulation on either or both cotyledon surfaces, tissue

necrosis. 7 : Abundant to heavy sporulation 1ainly on lower surface, light to scattered

sporulation on upper surface, tisue necrosis and chlorosis may be present. 9 = Heavy sporulation on lower surface, none to light sporulation on upper surface,

slight to no tissue necrosis. Chlorosis eay be present.

2

... •• -·HYPERSENSITIVE < :~ - NECROSIS

9 ~ (ixj)

Disease Index: DI= ~i=.li:...O __

~""z:: TISSUE ""' NECROSIS

n . n=total plants, i=IP class, j:number plants/class.

~~J = SPORULATION yyY

Selection: Susceptible controls, IP=7-9 by 7 days PI; partial resistance, IP=3-5; resistant plants, IP=0-1.

HOST RECOVERY: Selected plants uy be grown· on in the 111ultipots until requu1ng transplanting, or vernalized and flowered. Cotyledons of susceptible and partially resistant plants should be removed to prevent the spread of inoculua and movement of the fungus systemically into the plants. Plants say be rid of PP by spraying or dropping 'Ridomil' systemic fungicide, 0.1 g/liter on infections.

RESISTANCE A wide range of host specific variation exists in PP. Organism appears to be adaptible to numerous hosts other than that on which it occurs naturally (Dickinson and Greenhalgh, 1977). 1. 8. oleracea, cc: Little effective R exists. Monogenic dominant race specific R is

in cc. i P. I. 1:3902:3 and cc .c P. I. 245015 (Natti et. al., 1967), cc .c cv. 'January King' and wild cc, contains resistance (Greenhalgh and Dickinson, 1976).

2. Resistance in cotyledons does not ensure resistance in other parts. Sources in cc.i, R. L. Gabrielson, CrGC.

3. aa: oligogenic R exists in aa.p (Leung, 1991), source CrGC. 4. Race specific resistance can be found in a number of open pollinated crucifers. 5. There is a great need to identify race nonspecific resistance to PP.

MULTIPLE PATHOGEN INOCULATION Plants may be inoculated on one half of a cotyledon with one race of PP and on other halves with other races. Hark initial inoculation with needle puncture on margin of cotyledon, then proceed clockwise with other races· or additional pathogens.

3

Plants may be inoculated first on roots with FOG, PB or AR, then on cotyledons with PP, AG or LH. Multiple resistant survivors may later be inoculated with TUH, XGG, EG and EP. AG and PP inoculu• may be mixed, then applied. Interactions among host responses may occur depending on host and pathogen genotypes. For dual PP and AG inoculations, infectional incubation temperature should be 16 c.

APHANOM'ICES RAPHANI

ALBUGO CANDIOA

AIJERNARIA BRASSICICOLA

RHZOCTONIA SOLAN I

ERWINIA CAROTC't'aiA

TuloiV

OA't'S 0 7 14 21

H··················=·= .... ·········=·=·=~T:r S:·=·=·=·=·=·:·=·:·.···=·=·=·:·:·:·.·.·.;

... Q t---t !:::;:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;J

~i::::::;::l~:j:;:;:;:;:;:;:;:;::f 24'C 24'C

28 3S I

l---!.___ ____ l_.....:·:,;w;·:·:·:,;w;Jfeo;w;·:·:·~,;w;·:·:·;;w;:·:·:·;;w;:·:·:·;:;w~:·:·:1

... Q 1--i:=:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::@

1---1'-.....----;:;]F.;::::"-!:::"-::::~:::::~::~~::::~:::::"-::::"-::::"-::::·~.?.~::::-~::::lii:::::~::::~::::l

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4

16'C III'C

~ INnR.tCTloN PHENOTYPE

~ • 100 S RH

IN<:UIIATOA

~ I

DOIIIIJ Nildl!ll

Peronospora parasitica Pers. ex Fr. (PP)

ORGAN I Sit Obligately biotrophic intercellular parasite with large globular to coarse lobed haustoria. Hastigo;ycotina, Ooaycetes, Peronosporaceae. Air-borne conidia, soil-borne oospores. Leaf, ste1, root, flower invading.

LIFE CYCLE

APPEARANCE Macroscopic: Fine dense to loosely scattered white 'furry' 1at of conidiophores, usually

on lower surface of leaves under chlorotic or partially necrotic lesions. Oosporangial galls in ste1s and flowers may be light colored.

Nicroscopic: Hvceliul in host tissues, intercellular coenocytic, with large globular to much branched lobulate haustoria, hyaline. Conidiophores emerging singly or in groups through stomates, dichotoaously branched with clusters of ter1inal conidia which are easily detached by changes in RH and air aovement. Oospores produced in senescent tissues, thick s1ooth epispore wall, evidence for germination of oospores is lacking.

Variation (Dickinson and Greenhalgh, 1977) Stability: Little known about stability of existing variation in PP, though gene­

specific variants appear to arise easily. Kechanisas: PP is reported to be heterothallic (de Bruyn, 1937; HcHeekin, 1960). Phenotypic: Pathotypic variation (Greenhalgh and Dickinson 1975). A wide range of host

specific variation exists. PP appears to be able to grow on a range of hosts other than the one from which it was collected. Specificity exists also for tissue type attacked, roots have less specificity for isolates than leaves and cotyledons (HcHeekin, 1969). Pathotypes from B. oleracea, cc, B. juncea, aabb, B. ca11pestris,

5

aa, and radish, rr, and other wild species exist commonly. Gene-specific pathotypes race-1 and race-2 have been described for cc (Hatti et. al., 1967). See Resistance section.

INOCtR.UK Initial: Fresh sporangia collected from freshly sporulating lesions, PP does not store. Propagation: Produce inoculum on susceptible host grown, inqculated, and incubated as

described above. Conidia are collected by very gently removing (cutting with sharp blade) the cotyledon while holding it firmly with forceps. Cotyledons are placed in a 10-25 al beaker containing a few ml of distilled water. With a Pasteur pipette and bulb, gently wash off conidia in a streu of water sucked up and expelled fro11 the tip of the pipette. Discard cotyledons and quantify conidial suspension.

Quantification: Using a hemocyto1eter, count spores and adjust to appropriate concentration using distilled water.

For1ulation: Glass distilled water suspension of freshly collected conidia at 104 -105

conidia/a!.

STORAGE PP will not store satisfactorily apart from host tissue, oospores have not been germinated. Conidia and sporangia of other oo1ycetes have been stored at -20 C for varying periods of time either collected in vials or on frozen host tissue. Hore research on storage of PP is needed. TetPOrary Maintenance: Inoculated cotyledons may be excised 12-24 hr after inoculation

and their petioles inserted in 1% water agar (BWA) containing 10uH benzyladenine (a cytokinin to maintain cotyledon green) in small plastic boxes 'Baby-Boxes'. PP can be maintained on BWA for more than 2 weeks in a lighted growth cabinet maintained at 4-12 C. Baby-Boxes are useful for the transport of PP isolates.

6

~ I

(.,.......

,'flll""eo.. i

SOURCES CrGC (various, pathotypes), R. L. Gabrielson (seasonally).

REFERENCES de Bruyn, H. L. G. 1937. Heterothallis1 in Peronospora parasitica. Genetica. 19:553-

558. Dickinson, C. H. and F. R. Greenhalgh. 1977. Host range and taxono1y of Peronospora on

crucifers. Trans. Br. Hycol. Soc. 69:111-116. Greenhalgh, J. R. and C. H. Dickinson. 1975. Differential reactions of three crucifers

to infection by Peronospora parasitica (Pers. ex Fr.) Fr. Phytopathol. Z. 34:131-141.

Greenhalgh, J. R. and C. H. Dickinson. 1976. Studies of crucifer seedling resistance to Peronospora parasitica. Ana. Appl. Bioi. 84:278-281.

Humaydan, H. S. 1974. Hultiple disease resistance and genetics of Raphanus sativus. Ph.D. Thesis Univ. of Wisconsin, Hadison. 136 pp.

Leung, H. 1981. Breeding for aultiple disease resistance in cytoplasmic male sterile Brassica campestris L. H.S. Thesis, Dept. of Plant Pathology, University of Wisconsin, Hadison. 73 pp.

Lin, c. Y. 1981. Studies on downy mildew of Chinese cabbage caused by Peronospora parasitica. pp. 105-112. InN. S. Talekar and T. D. Griggs, (eds), Chinese Cabbage. Asian Veg. Res. and Dev. Center, Shanhua. +89 pp.

HcHeekin, D. 1960. The role of the oospores of Peronospora parasitica in downy aildew of crucifers. Phytopathology. 50:93-97.

HcHeekin, D. 1969. Other hosts for Peronospora parasitica fro• cabbage and radish. Phytopathology. 59:693-696.

Natti, J. J., H. H. Dickinson and J. D. Atkin. 1967. Resistance of Brassica oleracea varieties to downy aildew. Phytopathology. 57:144-147.

Yerkes, W. D. and C. G. Shaw. 1959. Taxonomy of the Peronospora species on Cruciferae and Chenopodiaceae. Phytopathology. 49:499-507.

7

CRUCIFER GENETICS COOPERATIVE DEPT. OF PL~NT P~THOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, H~DISON, WISCONSIN 53706 (608-262-6496)

WHITE RUST

Albugo candida (Pers. ex. Hook.) Kuntze (AC)

HOSTS Cruciferae, Capparidaceae (Hukerji, 1975).

DISEASE Seed borne, wind and rain spread. White spreading blisters (sori) on leaves, stems and inflorescence. Frequently accoapanied by hypertrophy and galls. On inflorescence commonly causing 'stag heads' and phyllody. Often found in association with Peronospora parasitica, (Bains and Jhoaty, 1985). Infected parts senesce prematurely except for isolated leaf lesions which may form 'green islands'.

DISEASE CYCLE 1° Inoculum oospores in ~ soil an-d on seed.

Germination in water, Premature.~ zoospore release. senescence and ~ \ death of tissues. Zoosporang1a "

t on plant parts. Invasion of

! · stoaata by Production of gera tubes. oosporangia in Release of I meriste1 tissue, zoosporangia ~ hypertrophy, to air. Intercellular galls. f colonization of

leaf and stem

HOST PROPAGATION

Production of parenchyu by zoosporangial sori, ~ haustoria. white blisters under epidermis.

Seed sown 0.5 em deep singly in individual pots of 12-pack multipot in 'peat-lite' at 24 C, 13-24 hr, 25~flol s· 111·z for 5 days or until cotyledons first expand. Irrigate with 0.5 x Hoagland s daily to run off.

TARGET TISSUE Cotyledons, 24-48 hr after expansion.

1

DSAC 05-13-85 WILP~U

INOCIUTION Place a Spl drop ·of zoospore inoculu1 on each 1/2 cotyledon using micropipette. Prevent droplets fro1 evaporating. Gently agitate inoculu1 and keep it cool to maintain zoospore motility.

INCUBATION Infectional Environ1ent: Maintain inoculum droplets on cotyledons 12 hr, 20 C, 100% RH.

Place plants in dew chamber in dark.

Incubation Environ~ent: Same as host propagation environment. Grow plants for 7 days.

EVALUATION Observe interaction phenotype (IP) on cotyledons 7 days after inoculation (post­inoculation, PI). Interaction Phenotype: Symptols and signs range from minute necrotic, hypersensitive

flecking under inoculu• drop to increasing numbers of pustule on the adaxial (upper) surface, then increasing numbers of pustules on the abaxial (lower) surface of the cotyledon. Highly compatible types have little sporulation on upper surface and large coalescing, spreading pustules on lower surface of cotyledons. Hore or less chlorosis around pustules 1ay occur. Compatible interactions can be accompanied by epinasty of the cotyledon. Invasion may become systemic.

Scale: Plants scored 0-9 at 7 days PI. 0 = No symptoms on either leaf surface. 1 : Saall, pinpoint to larger brown necrotic flecks under inoculation point on upper

surface, occasionally necrosis extending to lower epidermis. No sporulation. 3 : Very sparse, 1 - few, ainute scattered pustules on upper surface, none to very

few pustules on lower surface. 5 : Few to many scattered pustules on upper surface. None to few scattered pustules · on lower surface.

7 = Hany to few pustules on upper surface. Hany scattered small to larger pustules on lower surface.

9 : Very few to no pustules on upper surface. Hany large coalescing pustules on lower surface.

2

r 0 \ 3 5 1

9 2: (ixj)

Disease Index: DI= £-i=.::...D __ n

n=total plants, i=IP class, j:nu1ber plants/class. Selection: Susceptible controls, IP=7-9 by 7 days PI; partial resistance, IP=3-5;

resistant plants, IP=0-1.

HOST RECOVERY Selected plants aay continue to be grown in the aultipots until transplanting. Cotyledons of susceptible and partially resistant plants should be reeoved to prevent spread of sporangia and aoveaent of fungus systemically into plant.

RESISTANCE A wide range of host specific variation exists in the pathogen. Effective resistance exists in so1e lines and cultivars of most crops. Resistance is largely unexplored. Resistance in cotyledons does not necessarily assure resistance in the remainder of plant. Susceptibility to PP 1ay predispose resistant plants to AC. 1. R. sativus, rr .r: Host cvs. susceptible to AC race-1. Honogenic doainant

resistance. Ac1/Ac1 found in cvs. 'China Rose Winter' and 'Round Black Spanish' (Williaas and Pound, 1963).

2. B. juncea, aabb: Host cvs. susceptible to AC race-2. Oligogenic resistance in P.I. 347618. Honogenic dominant.

3. B. caapestris, a: Honogenic doainant gene Ac2/Ac2, confers resistance to AC race-2 (James and Willia1s, 1980).

4. Ac2/Ac2 for resistance to AC race-2 in B. nigra (Delwiche and Williams, 1981). Polygenic for•s of resistance exist in B. campestris (Edwards and Williaas, l'JSS).

SOURCES P. H. Williams

KULTIPLE PATHOGEN INOCULATION Plants aay be inoculated on one half of a cotyledon with one race of AC and on other half with other races. Hark initial inoculation with needle puncture on margin of cotyledon, then proceed clockwise with other races or additional marker holes.

3

9

Plants may be inoculated first on roots with FOC, PB or AR, then on cotyledons either AC, PP, tH, or AB. Hultiply-resistant survivors 11ay later be inoculated with TUH, XCC, EC, and EP. AC and PP inoculum may be mixed, then applied. Interactions among host responses 11ay occur depending on host and pathogen genotypes.

/~ /

r NHITE RUST

Albugo ciUidida (Pers. ex. Hook.) 0. Kuntze

ORGAN I Sit Obligately biotrophic intercellular parasite with globular haustoria. Hastigomycotina, Ooaycetes, Albuginaceae. Airborne sporangia, soil and seed borne oosporangia. Leaf, stea and flower invading.

LIFE CYCLE (Hukerji, 1975)

0 .... ,.,.., . '2 oeJp•r•wt., ''-""

~~ ~ ... ,. ...

APPEARANCE Kacroscopic: Hasses of hyaline sporangia produced under lower and upper epidermis

appears as white shiny blisters in aass. Asexual sporangia are a white to creamy white powder, oosporangial galls after host tissue has necrosed may show olive to brown color of oosporangia.

Microscopic: Hyceliua: Intercellular coenocytic. Haustoria: Small, globose, many per cell. Sporangia: Hyaline, globose to oval, 12-18p diaaeter in basipetal chains fro• clavate

sporangiophores 30-45 x 15-18p.

5

Oospores: Globose 30-55~, chocolate-olive brown, epispore thick, verracose to tuberculate or with low blunt ridges.

Zoospores: Biflagellate heterokont 4-8 emerging from sporangium, actively motile, then encysting and germinating with germ tube in a few minutes.

~ """ ~ "Z.oospor•

/fiOO )(

VARIATION (Pound and Williams, 1963) Stability: Little known about stability of existing variation in A. candida. "echaniSIS: A. candida may be heterothallic (Sansoae and Sansoae, 1974). Phenotypic: Pathotypic variation. A wide range of host-specific variation exists in A.

candida. Pathotypes, races, generally best adapted to host species on which they occur naturally or in the crop. Natural or primary hosts for races (Pound and Williams, 1963). Race-1, Raphanus sativus; race-2, Brassica juncea; race-3, Armoracea rusticana; race-4, Capsella bursa-pastoris; race-S, Sisytbriu• officinale; race-6, Rorippa

islandica; race-7, B. catpestris; Sinapis alba appears to be a 'universal' suscept.

INOCUlllll Initial: Sporangia from frozen storage. Propagation: Produce inoculum on 5-7 day-old cotyledons of 1° host as described herein.

Collect fresh sporangia from 8-12 day, PI, infections of IP=7-9. Using a cyclone spore collector with 100 gelatin capsules, rupture sporangia! sori and collect fresh sporangia.

Zoospore Production: Add a few 19 of freshly collected or frozen stored sporangia to 5-15 tl distilled water in a 25 ml erlenmeyer flask. Cover flask with 'Para fila', shake vigorously to suspend sporangia and incubate 2-3 hr at 12-16 C. Zoospores will eaerge and reaain motile in 1-2 hr.

6

Quantification: Keep zoospores cool, 4-12 C, in ice water bath to maintain their motility. Using he1ocyto1eter, quantify zoospores/mi. Prior to placing cover glass on spore counting slide, hold a saall piece of cotton, '0-tip', soaked in formalin near drop of zoospores for a few seconds and zoospores will stop loving.

l s ~~~ ,,,:._, \ ' ( r.,- " ~ ,,_ ' ,, ~-+.) rJ·· 0 \~-.._..... \ ;__)vV ':7 i ' -') f~ CJ f \,., \)\/' Q I' !---.

ForiUlation: Glass distilled water suspension of motile zoospores. Optional concentration 105 spores/11. Use pre-chilled water to make necessary dilutions. Use of motile zoospores ensures viability of inoculua. Do not use ungerainated sporangia as inoculu1.

STORAGE Store sporangia in 100 gelatin capsules in glass screw-cap vials at -20 C. Longevity of sporangia in storage varies fro• a few months to 1ore than 2 years. Be sure not to thaw and refreeze sporangia in capsules not used for immediate inoculum.

SOURCES CrGC (AC races-1,-2,-7). Hajor national collections of fungi do not store AC.

REFEREHCES Bains, S. S. and Jhoaty, J. S. 1985. Association of Peronospora parasitica with Albugo

candida on Brassica juncea leaves. Phytopath. Zeit. 112:28-31. Delwiche, P. A. and P. H. ·williams. 1981. Thirteen marker genes in Brassica nigra. J.

Heredity. 72:289-290. Ja1es, R. V. and P. H. Williams. 1980. Clubroot resistance and linkage in Brassica

ca•pestris. Phytopathology. 70:776-779. Hukerji, K. G. 1975. Albugo candida. CHI Descriptions of Pathogenic Fungi and

Bacteria. No. 460. Coaaonw. Hyco!. Inst., Kew. Pound, G. S. and P. H. Williams. 1963. Biological races of Albugo candida.

Phytopathology. 53:1144-1149. Sansoae, E. and F. W. Sansome. 1974. Cytology and life-history of Peronospora

parasitica on Capsella parsa-pastoris and of Albugo candida on c. bursa-pastoris

7

and on Lunaria annua. Trans. Br. Hycol. Soc. 62:323-332. Verma, P. R. and G. A. Petrie. 1975. Germination ofoospores of Albugo candida~ Can.

i. Bot. 53:836-842. Verma, P. R. and G. A. Petrie. 19SO. Effect of seed infestation and flower bud

inoculation on systemic infection of turnip rape by Albugo candida. Can. J. Plant Sci. 60:267- .

Williams, P. H. and G. S. Pound. 1963. Nature and inheritance of resistance to Albugo candida in radish. Phytopathology. 53:1150-1154.

8

/~ /

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HADISON. WISCONSIN 53706 (608-262-64961

BLACK ROOT

Aphanotrces raphani Kendrick (AR)

HOSTS Cruciferae including Raphanus, economic brassicas, and other wild species (Humaydan and Williams, 1975: Kendrick, 1927)

DISEASE

DSAR 06-22-87 WILPAU

Soil-borne. Darkening, blackening of invaded primary and secondary roots and hypocotyl, black, shrunken, constrictions, scurfy appearance of radish bulbs, particularly severe on cv. 'White Icicle'. On red globes often accompanied by purpling of red pigment, roots severely distorted by arrested growth. Occasional invasion .of seedling hypocotyls, petioles, cotyledons and true leaves, accompanied by black streaking and yellowing of leaf or cotyledon.

DISEASE CYClE

HOST PROPAGATION

1° inoculum ~ oospores in soil.~

Oospore production ~ in cortical and root tissues.

f ~ Necrosis of invaded tissues.

Zoospore release.

Intercellular colonization.

Zoospores.

2° root emergence.

1. Seed sown 2 mm apart 0.5 em deep in clean washed sand. 20 C, 250pmol s-lm- 2 , 18-24 · hr.Irrigate with 0.5 x Hoagland solution, until cotyledons expand.

2. Alternative: Seed sown on slanted moist filter paper. 20 C, 250pmol s-lm- 2, 18-24 hr.

TARGET TISSUE Seedling roots. Lift seedlings with spatula. Dip roots in water to remove excess sand. Blot excess water on towelling.

INOCULATION Roots and hypocotyls from sand or filter paper grown seedlings are immersed for 1 hour in zoospore inoculua in a 50 ml beaker, then transplanted singly to multipots in peat-lite.

IHCUBATIOH Infectional Environment: Haintain host propagational environment. 24 C, 13-24 hr,

250pmol s-lm- 2 • Irrigate daily to run-off with 0.5 x Hoaglands. rt is important to maintain field capacity moisture during the incubational period of 3-4 weeks. Additional daily irrigation with water aay be necessary.

Incubation Period: Grow plants for 3-4 weeks post inoculation (PI).

EVALUATION Lift plants, shake off excess soil, wash roots thoroughly for viewing. Interaction Phenotype (IP): Symptoms range from a damping off starting 6-8 days

afterinoculation, to severely blackened distorted mature roots with scurfy outer tissue and black to grey inner tissue. Resistant roots show no symptoms to small superficial scurfy lesions usually at point of 2° root emergence.

Scale: Plants scored 0-9, 3-4 weeks after inoculation 0 = No symptoms, normal root development. 1 = Few, very small superficial, scurfy lesions at points of 2° root emergence. 3 = Small surface lesions at points of 2° root emergence, normal root size. 5 = Slowly developing scurfy lesions scattered on roots, some lesions slightly

shrunken, normal root size. 7 = Deeply shrunken constrictions and deformities, large scurfy lesions, black tissue

under lesion, some root and top stunting. 9 = Severely infected, blackened roots, sometimes damping-off prematurely, root

enlargement and top growth restricted, plant death.

3 7 9

9 2::_ (ixj)

Disease Index: DI= i=O :.....;:. __ n

n=total plants, i=IP class, j:number plants/class. Selection: Susceptible plants, IP=?-9; partial resistance, IP=3-5; resistant plants,

IP=0-1.

HOST RECOVERY Selected survivors may be transplanted to pots, vernalized and flowered. Partially resistant or susceptible plants may be rid of AR by dipping roots in 0.1 g 'Ridomil' fungicide/liter.

2

RESISTANCE Little is known about pathotypic variation of AR, most common commercial red globe and white radishes are susceptible. Cv. 'White Icicle' is highly susceptible. Turnips, especially 'Purple Top White Globe', are susceptible. A range of variation in resistance to AR exists in many radish and brassica cvs. Resistance in radish is oligogenic and highly heritible (Humaydan et. al., 1976). Radish cvs. 'Fuego' (Northrup King Seed Co.), 'Far Red' (Northrup King Seed Co.), and 'Fancy Red' (Harris-Horan Seed Co.), are AR resistant, some oriental types of radish are resistant. Turnip line PTJR (Harris-Horan Seed Co.) is resistant to AR, PB, and RS.

HULTIPLE PATHOGEN INOCULATION Plants may be first inoculated with AR as described, then dipped in FOG and transplanted. 24-43 hr later they may be inoculated on the cotyledons with AG, PP, AB or LH. Survivors may later be inoculated with RS, XGG, TUH or EG. All combinations have not been examined, but no interactions appear to exist between FOG-R, AG-R and AR-R genotypes to render plants more or less resistant than when inoculated singly. AR-S and FOG-S types die more rapidly in dual inoculations (Humaydan, 1974).

3

BlACI ROOT

Aphanoayces raphani Kendrick (AR)

ORGAHISH Intercellular, necrotrophic parasite. Hastigomycotina, Oomycetes, Saprolegniaceae. Soil-borne oospores, water-borne zoospores. Root, hypocotyl, rarely leaf invading.

LIFE CYCLE (Kendrick, 1927)

.... ,.--.

APPEARANCE In Culture: On RA or PDA, a .dense white to cream color with little aerial mycelium. Hicroscopic:

Mycelium: Hyaline nonseptate, thick, 8-llu moderately branched. Hany much branched short side hyphae from which zoosporangia form.

Zoosporangia: Extensions of hyphae as slightly tapering coiled and twisting discharge tubes for cytoplasm.

-z.•sp•"• ~ ~ . So•':.:;:

' . .

~ •-.'tf..CAJ,M.....,

Zoospores: Biflagellate, heterokont. Emerging from zoosporangial cysts which form in clusters at tip of zoosporangial discharge tube. Zoospores 10.2u diameter.

· Oospores: Produced in tissue and in RA after 7-10 days at 28 C. Spherical 18.1-30. 3u (avg. 23.3u), smooth, thick, hyaline wall. Produced in oogonia 24.2-43.6u diameter. ·

4

VARIATION Stability: No loss of pathogenicity after 2 years of periodic transfer and storage at

4C. HechanisiS: AR is homothallic, oogonia and antheridia produced on same thallus. Phenotypic, Pathotypic: No pathotypes have been reported in AR. Wide host range among

crucifers, 15 noncrucifer families tested were not infected (Humaydan, 1974).

IHOCULUft Initial: Hyphal tips from RA cultures or freshly isolated from infected seedlings used

to bait from long-term oospore storage, see Storage Retrieval. Propagation: From actively growing margin of culture on RA, 24 c, transfer 5 mm discs

singly to 25 ml radish peptone broth, RPB, in 125 ml flasks. Incubate flasks 24 c for 2 days. Transfer, aseptically, mycelial mat to 25 ml of sterile distilled water (pH=4-5.3) in a petri plate. Incubate plates 24 hr at 20 C. Zoospores will be released and motile.

:rf&,·l .. clr IIIII#'./ ..,.,.,. .z.,lc.-,aol:'

Quantification: Using hemocytometer, count zoospores/mi. Prior to placing cover glass on counting-slide, hold a small piece of cotton, rQ-tip', soaked in formalin near drop of zoospores for a few seconds, zoospores will stop moving.

For1ulation: Adjust inoculum to contain 104 zoospores per ml of glass distilled sterile water, use zoospore suspension directly to dip seedling roots in.

STORAGE Short Ter1 (IOnths): On RA slants under oil at 4 C, fungus remains viable for 5 months

(Humaydan, 1974). Long Ter1 (years): As oospores, dried in soil or RA at 4 Cor -20 C (Ghafoor, 1964).

Grow cultures on RA plates 3-4 weeks at 24 C by which time abundant mature oospores will have been produced. Cut agar and mycelial mat into 0.5 em squares and let agar dry at 32 C for 3-4 days. Store dried agar and oospores in vials or mixed with sterilized sandy loam in vials at 4 C or -20 C.

5

Retrieval Fr01 Storage - Baiting Hethod: Soak oospore-agar-squares in flasks of distilled water for 3-4 days with daily changes of water. Bury 2-3 soaked oospore­squares in the center of each pot of a 12-pack at a depth of 2 em in peat-lite and sow 3 radish cv. 'White Icicle' or turnip cv. 'Purple Top White Globe' in the center of each pot at a depth of 0.5 em. Grow seedlings in the same environment as host propagationfor 2-3 weeks. Ten days after seeding, thin seedlings to 1 plant per pot. A few oospores will germinate and infect the 'bait plant' roots in 10-20 days. Keep soil moisture high during the baiting period. After 3 weeks remove plants, wash roots thoroughly, examine them for AR infection. Surface sterilize lesion areas and place small cubes of infected tissue on streptomycin-benomy l radish agar (SBRA). Transfer hyphal tips of AR to RA, incubate at 24 C.

SOURCES - PHW

REFERENCES Ghafoor, A. 1964. Radish black-root fungus: host range, nutrition and oospore

production and germination. Phytopathology. 54:1167-1171. Humaydan, H. S. 1974. Hultiple disease resistance and genetics of Raphanus sativus. PhD

Thesis, Univ. of Wisconsin, Hadison. 136 pp. Humaydan, H. S. and P. H. Williams. 1975. Additional cruciferous hosts of Aphanomyces

raphani. Plant Disease Reptr. 51:113-116. Humaydan, H. s. and P. H. Williams. 1978. Factors affecting in vitro growth and

zoospore production by Aphanamyces raphani. Phytopathology. 63:377-331. Humaydan, H. S., P. H. Williams, B. J. Jacobsen, and H. L. Bissonnette. 1976.

Resistance in radish to Aphanomyces raphani and Rhizactania salani. Plant Disease Reptr. 60:156-160.

Kendrick, J. B. 1927. The black-root disease of radish. Indiana Agr. Expt. Sta. Bull. 311. 31 pp.

6

~·

!" i

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN 53706 (608-262-6496)

HOSTS

BLACKLEG

Leptospbaeria 1aculans (Desl.) Ces. et de Not. (LH) Pho.a linga1 (Fr.) Tode

Cruciferae, especially Brassica oleracea, cc; B. campestris, aa; B. napus, aacc; also on various other genera (Punithalingam and Holliday, 1972).

DISEASE Seed-borne, wind, rain and plant debris spread. Pale lesions on cotyledons, leaves and stems. Lesions become greyish with dark pycnidia at enter. Lesions often have dark or purplish margins, developing into dry girdling cankers at soil level or higher on stem, girdled plants die. A dry rot on stored heads and roots.

DISEASE CYCLE to Inoculum:

~ / Hycelium in seed plant debris.

Seed pod infection.

I Ascospores.

Systemic movement Perithecia in flower on dead stems. stalk?

~ \ Girdling of stem, Q death of plant.

Infection of ~ leaves, stems, flowers, ~ cankers, pycnidia.

HOST PROPAGATION

Pycnidia pycnidiospores on seedlings.

~ Spread in seed bed.

I Transplants.

Seed sown 0.5 em deep singly in individual pots of 12-pack multipot in 'peat-lite', 24 C, 13-24 hr, 2SOpmol s· 1m· 2 for 5 days or until cotyledons first expand. Irrigate with 0.5 x Hoaglands solution daily to run off.

DSLH 06-22-87 WILPAU

TARGET TISSUE Cotyledons, 24-48 hr after expansion. INOCULATION Using a 10 micro liter pipette, place a lOul drop of inoculum, pycnidiospore suspension 6n each half cotyledon. After the inoculum drops are deposisted, puncture the drop and cotyledon with a single prick using a clean needle or pin. Hore than one isolate can be inoculated by first puncturing the margins of each half cotyledon with a clean pin then depositing in each marked half cotyledon a different isolate. Use a separate needle to puncture wound with each isolate.

INCUBATION Infectional Environ1ent: No special environment needed, permit inoculum drops to

evaporate under host propagational environment. Incubation Environ1ent: Same as host propagation environment. Grow plants 10-12 days.

EVALUATION (Delwiche, 1980) Observe interaction phenotypes (IP) on cotyledons 10-12 days after inoculation. Interaction Phenotype: Symptoms first appear 6-8 days PI as limited necrotic tissue

around the wound in resistant phenotypes and as a faint bleaching or sheen of a diffuse area around wound in susceptible IP's. IPs are assigned according to lesion size, amount of tissue blackening and presence of tissue collapse and sporulation.

Scale: Plants scored 0-9 10 days after inoculation. 0 : No darkening around wound, as in controls. l : Limited blackening around wound, lesion diameter = 0.5-1.5 mm, faint chlorotic

halo may be present. Sporulation absent. 3 = Dark necrotic lesions, l.S-3.0 mm, chlorotic halo may be present, sporulation

absent. 5 = Non sporulating 3-6 mm lesions, sharply delimited by dark necrotic margin, may

show -grey-green tissue collapse as in IP 7 and 9 or dark necrosis throughout. 7: Grey-green tissue collapse 3-5 mm diameter, sharply delimited, non

darkened margin. 9 = Rapid tissue collapse at about 10 days, accompanied by profuse sporulation in

large, more than 5 mm, lesions with diffuse margins .

x WOUND

li.) CHLOROSIS

~ DARK NECROTIC TISSUE

,~, GRAY-GREEN TISSUE '"-' COLL~SE

. : •; ~YCNIDIA

0 SHAR~ DARKENED MARGIN

Q SHAR~ NON-DARKENED MARGIN

r Notes:

1. Occasionally if cotyledons contact soil, senesce under humid conditions, or are observed more than 14 days PI, sporulation may be seen in IP=S.

2. If cotyledons are wounded and inoculated directly on vein, lesions of any IP=l-9 may extend rapidly down petiole and invade hypocotyl.

9 ~(ixj)

Disease Index: DI = i=O ~~--

n

n=total plants, i=IP class, j:number plants/class

Selection: Susceptible, IP=7-9; partially resistant, IP=S; resistant, IP=O, 1 and 3.

HOST RECOVERY Selected plants may be grown in the multipots until requiring transplanting. Cotyledons of susceptible or partially resistant plants should be removed to prevent sporulation and/or systemic spread into plant. Susceptible plants may be sprayed or drenched with Benomyl fungicide, 0.2 g/liter to disinfect the plant.

RESISTANCE A wide range of pathotypic variation exists within pathogen, resistance exists in some crops to some pathotypes, resistance largely unexplored. Resistance in cotyledons does not necessarily assure resistance in other parts. 1. 8. aleracea, cc: Largely susceptible to cc strains. 2. B. campestris, aa: Largely susceptible, resistance unexplored. 3. B. napus, aacc: Largely susceptible to aacc strains of LH, some race specific

cotyledon resistance has derived from aacc.r cvs. 'Jetneuf' ,'Quinta' and'Primor', linked dominant monogenes Lml and Lm2 (23.5!3.7%) are in French lines R39 and cv. Girita, respectively (Delwiche, 1980) source (CrGC).

HULTIPLE PATHOGEN INOCULATION Plants may be inoculated with various isolates of LH as described or inoculated first on the roots with FOC, AR or PB, transplanted and inoculated on the cotyledons with LH, AC, PP or AB. Hultiresistant survivors may later be inoculated with fUH, XCC, EC, and EP. Interactions have not been explored.

3

ORGAN ISH

BLACKLEG

Leptosphaeria taculans (Des•.) Ces. et de Note. (LH) Phota linga1 {Fr.) Tode.

Facultatively saprophytic, necrotrophic pathogen. Ascomycotina, Sphaeriales, heterothallic. Seed-borne mycelium, air-borne ascospores, rain-splashed pycnidiospores, leaf, stem, seed, root invading.

LIFE CYCLE (Kendrick, 1927)

.; flll4 ...... .., ~t- t.-.,. ... 4

APPEARANCE

'·~·' ft .....

••••

In Culture: Highly variable in rate and type of growth, pycnidial production and degree of pigmentation. On oatmeal agar mycelial mat is white, green, or yellow brown tinges. Host isolates sporulate abundantly on V-8 juice agar exposed to light. Pycnidial exudate often red to amethyst or white.

Hicroscopic: Mycelium: Septate, pigmented or hyaline. Pycnidiospores: Aseptate, biguttulate, hyaline, shortly cylindrical, 1-2 x 3-5u.

Borne in pseudo-parenchymatous (thin-walled) or pseudo-sclerenchymatous (thick­walled) pvcnidia. 200-600u diameter.

Ascospores: Biseriate, 5-septate, cylindrical to ellipsoidal, ends rounded, guttulate, 35-70 x 5-~, at maturity yellow-tan, borne in cylindrical, a-spored,

· bitunicate asci within erumpent, pseudosclerenchymatous, globose black pseudothecia 300-SOOp diameter.

"'1'\.!J.Mn.......- f ... - •. fHc.t~""

4

VARIATION Stability: Highly unstable in culture, numerous sectors, frequently to mycelial, non

sporulating types of lower pathogenicity, stability in nature, unknown. Hechanisas: Heterothallic, bipolar (+,-), in culture, ascocarp production on wheat straw

or B. napus residue-agar, requires short wave length light (Delwiche, 1980), difficult to obtain ascospores in culture.

Phenotypic: Isolates generally classified into strongly pathogenic (SP) and weakly pathogenic (WP) strains. WP isolates grow more rapidly than SPs on V-8A and MEA and produce a yellow-brown, water soluble pigment on COY. Radial growth of WP isolates is only partially inhibited on PDA + 0.02% lactic acid,whereas SPs were completely inhibited (Delwiche, 1980).

Pathotypic: Evidence for races on various host genera and species has been reported and is tentative (Petrie and Vanterpool, 1965; Thurling and Venn 1978; HcGee and Petrie, 1978; Pound, 1947). Pathotypes in rapeseed, aacc.o, based on cotyledon responses in 6 inbred differential lines (Delwiche, 1980), source of seed CrGC. Considerably more research is needed on pathotypic variation.

INOCUUJII Initial: Spores and mycelium from stored cultures. Propagation: Transfer stored cultures to V-SA slants or plate, incubate in light at 20-

24 C. When culture is sporulating actively, cut a 1x3 em strip of agar with pycnidia, and transfer to 10 ml sterile distilled water, sdw in test tube. Agitate tube vigorously on mixer to obtain a slightly cloudy suspension, approximately 106-107

spores/1111. Spread 0.5 ml of spor? suspension onV-SA plate. Incubate pla~e at 20-2AC~jn light. ]eeded-pijtes developdense mat of siiorulating pycnidla ·in 4-5 days. -i)

-Flood plates with 10 ml sdw, scrape surface to diScharge pycnidiospores. .

Quantification: Count spores with aid of hemocytometer, adjust concentration with sdw. Forsulation: Sdw suspension 107 pycnidiospores/ml .

. ~, ·' ~:~ . '.:'""· '/' ·,-. -.

5

,...._ .. ·~· . .:: · .. ·. ·-·

\ . .' ji'· ..

STORAGE Long Tera (years): Freeze drying, grow 4-5 day old cultures on V-BA, seed plates.

Mature pycnidiospore are harvested in 20% sterile skim milk. Scrape spores from surface to yield a suspension of more than 106 spore/ml. Dispense 0.2 ml spore suspension to sterile vials. Freeze, vacuum dry, seal, label, store at 4 Cor -20 C.

Short Tera (years- IDDths): On PDA under oil at 4 C in screw-cap vials.

SOURCES ATCC, CVS, CHI, R. L. Gabrielson, CrGC (cc, aacc isolates; mating types, pathotypes).

REFERENCES Delwiche, P. A. 1980. Genetic aspects of blackleg (Leptosphaeria maculans) resistance

in rapeseed (Brassica napus). Ph.D. Thesis, University of Wisconsin, Madison. 144 pp. McGee, D. C. and G. A. Petrie. 1978. Variability of Leptosphaeria maculans in relation

to blackleg of oilseed rape. Phytopathology. 68:625-630. Petrie, G. A. and T. C. Vanterpool. 1965. Diseases of rape and cruciferous weeds in

Saskatchewan in 1965. Can. Plant Dis. Surv., Can. Dept. Agric. Res. Branch. 45:111-112.

Pound, G. S. 1947. Variability in Phoma lingam. J. Agric. Res. 75:113-133. Punithalingam, E. and P. Holliday. 1972. Leptosphaeria maculans. CHI Descriptions of

Pathogenic Fungi and Bacteria. No. 331. Commonw. Hyco!. Inst., Kew. Thurling, N. and L. A. Venn. 1977. Variation in the responses of rapes (Brassica

napus and B. campestris) cultivars to blackleg (Leptosphaeria maculans) infection. Aust. J. Exp. Agr. Anim. Husb. 17:445-451.

6

CRUCIFER GENETICS COOPERATIVE DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN 53706 (608-262-6496)

FUSARIUI YEllOWS Fusaria oxrsporu. f. sp. conglutinaJJS (fOC)

HOSTS Cruciferae including Brassica oleracea, cc, and Raphanus sativus, rr, (Subramanian, 1970; Walker, 1968).

DISEASE Soil-borne vascular wilt favored by temperatures greater than 17 C. Progressive yellowing then brown necrosis of plant from lower leaves. Frequently unilateral, vascular browning. Plants stunted. Premature leaf drop.

DISEASE CYCLE 1° Inoculua in soil, ~

~ chlamydospores. ~

Survival in soil for years.

\ Colonization of necrotic tissues ~ conidia, chlamydospores.

HOST PROPAGATION

Death of plant.

Invasion of root tip and via wounds.

; Colonization of xylea vessels by hyphae, aicroconidia.

1. Seed sown 2 ma apart 0.5 em deep in clean washed sand. 20 C, 18-24 hr, 25~aol s· 1m· 2 • Irrigate with 0.5 x Hoagland's solution, until cotyledons expand.

2. Alternate: Seed sown on slanted moist filter paper. 20 C, 18-24 hr, 250paol s· 111" 2 •

TARGET TISSUE - Seedling roots. Lift seedlings with spatula, dip roots in water to remove excess sand. Blot excess water on toweling.

INOCULATION 1. Dip roots in inoculum, transplant 1 em apart in sand moistened with 0.5 x Hoaglands

solution. 2. Dip roots in inoculum, transplant singly to multipots in peat-lite.

DSFOC 05-13-85 WILPAU

IHCUBATIOM 14 days, 24 c soil, in soil temperature control tanks, 18-24 hr, 250pmol s- 1.-2•

Irrigate with 0.5 x Hoagland's.

INTERACTION PHENOTYPE {IP)

!..)1 J CO-' f,""

S"•; I 1-e""pc,.&n...,.<ft + ..... k.

SyaptOIS: Early progressive chlorosis of cotyledons and leaves, progressing to stunting, necrosis and death. Colonized roots brown to black, severely stunted. Vascular browning.

Scale: Plants scored 0-9 at 7-14 days. 0 = No sy1ptoms in tops or roots. I = Darkening of roots, no stunting or symptoms in tops. 3 = Darkening of roots; slight top stunting, no chlorosis. 5 = Dark stunted roots, tops stunted, slight chlorosis of cotyledons. 7 = Severe stunting of roots and tops, strong chlorosis. 9 • Severe stunting, necrosis, death.

0 3 5 9

9 2 (ixj)

Disease Index: DI= i=O :......:...--n

n=total plants, i=IP class, j:number plants/class. Selection: Susceptible controls, IP=7-9 by 14 days; partial resistance, IP=3-5;

resistant plants, IP=0-1, may be recovered ..

HOST RECOVERY Selected survivors may be transplanted to pots and grown at 20-24 C. If planting in field, roots may be dipped in a suspension of 0.2 g/liter beno1yl fungicide.

RESISTANCE (B. oleraeea var. eapitata, cc.c) 1. Konogenic dotinant: Foe/Foe. IP=0-1, breaks down at greater than 26 C. Effective

for more than 50 years in all regions. Widely available, (Asgrow Seed Co., Alf Christianson Seed Co., Ferry-Horse Seed Co., Northrup King Seed Co.).

2. Polygenic: Hore sensitive to temperature, breaks down at 22-26 C. Source, cv. Wisconsin Hollander 18 (Alf Christianson Seed Co., Keystone Seeds).

3. Susceptible: Cv. Golden Acre, various European cvs., e.g. Green Winter (Asgrow Seed Co., Alf Christianson Seed Co., Ferry-Horse Seed Co., Keystone Seeds). Sole broccolis (cc.i) and cauliflowers (cc.b) and other varieties are resistant to FOC.

4. Raphanus sativus, rr.r, resistance to FOC race-2 is polygenic and breaks at 23-25 C. Sources of R 'Red Prince' (Northrup King Seed Co., Harris-Horan Seed Co.), Scarlet Knight, Far Red (Northrup King Seed Co.), Fancy Red (Harris-Horan Seed Co.).

MULTIPLE PATHOGEN INOCULATION Plants inoculated with FOC may be simultaneously dipped in AR inoculate or inoculated on cotyledons with PP, AC, LH, or AB. FOC resistant plants may be inoculated later with PB, TUH, XCC, EC and EP or evaluated for TB.

3

FUSARIUit YEllOWS Fusariu• oJysporu f. sp. cmglutinans (FOC)

LIFE CYCLE Chlamydospores in soil and plant debris.

Germination. Chlaaydospores and aacroconidia. \

t Hypha! colonization of vascular tissue.

Hyphal colonization of necrotic tissue.

APPEARANCE (Booth, 1970; Walker, 1968)

/ Microconidia in vascular tissue.

In Culture: Myceliu• white, sometimes with purple tinge. Sparse then abundant, then floccose, beco1ing felted.

Kicroscopic, Kicroconidia: Abundant, variable, oval ellipsoid cylindrical straight to curved 5-12 x 2.2-3.5u, none to 3 septate.

Kacroconidia: Sparse, 3-5 septate, fusoid 27-66 x 3-Su.

Chla1ydospores: Abundant, saooth to ·rough walled, ter1inal and intercallary.

VARIATION

'' Sfj) ~~ n..J ~~~

Stability: Moderately variable in culture, little loss of pathogenicity in culture. Kechanists: Heterokaryosis, possible parasexual, no sexual stage. Phenotypic-Pathotypic: ·Races based on host specialization (Subramanian, 1970). Race 1: Mainly on B. oleracea, cc; cc.a, cc.a1, cc.b, cc.c, cc.g, cc.go, cc.i. Also

aa.o, aa.p, aa.r, aabb, aacc.r, rr, Lepidiu• sativum, Hatthiolis incana. Race 2: Mainly on R. sativus, rr, but also above hosts except cc.c, cc.g, and cc.b. Races 3 and 4: Hatthiolis incana cvs. Race 5: Saae as Race 1 abd attacks plants with monogenic dominant resistance (Type A).

4

IIIOCII.Uit Initial: Spores and ;yceliua fro1 stored cultures. Propagation: Add s1all amount of stored inoculu1 to PDA plate at 24 c.

growing hyphae to PDB in 125 11 flasks. Subculture

l. Shake at 20-28 C, 3-5 days, longer at lower te1peratures. 2. Still cultures may take 7-14 days.

Quantification: 1. Conidia: Filter culture through 2 layers cheesecloth, wash myceliua mat with SO

1l sterile water, combine filtrate and washing. Centrifuge at 10,000 rpe for 10 min. Resuspend spores in water and adjust concentration with he1ocytometer .

.s~t..r.. .. ~ c ... ,.,. .. ,.. .• ':•

- . ; ... !: :.

""'1 cck-.1 ;,.,_."f:s

.. ,, .... 5

2. Mycelial Fragments and Spores: Scrape washed 1ats into blender. Add SO •1 water/mat. Triturate 15 sec at high speed. Prepare 103, 104 , 105 , 106 , and 107

dilutions of •vcelial suspension. Pipette 1 •l of each concentration to tubes containing 9 11 of 0.25% water agar. Hix suspension well and transfer 1 1111 of each concentration to duplicate PCHB agar plates. Spread suspension evenly over plate using bent glass rod. Incubate 48-72 hr at 28 c. Count colonies at each dilution and estiaate nu1ber viable propagules.

5

For10lation: Water suspensions of spores, optional concentration = 108/al, or 1.5x104

~ycelial frag1ents. Spores and ayceliu• 1ay be co1bined. Satisfactory inoculu• can be prepared fro1 washed triturated aats at rate of 1 mat/50 al of water.

STORAGE(Alexander et. al., 1980; Tuite, 1969) Long Ter• (years): Cryogenic - lyophilization. Dry - in soil: add 0.5 al conidial

suspension fro• 3-day shake culture to screw cap vial containing 5 cc sterile loaa soil. Place vial at 28 C with loosened cap to perait slow drying. When dry, tighten cap. Store at 4 C or -20 C.

Short Ter1 (IDftths-years): On PDA slants, with or without aineral oil overlay.

SOURCES ATCC (races 1-4), CHI (races 1-4), CVS (race 1), CrGC (races 1,2,3,4;5).

REFEREMCES Alexander, H., P. H. Daggett, R. Gherna, S. Jong, F. Simone. 1980. ATCC Hethods I.

laboratory Hanual on Preservation Freezing and Freeze~Drying. ATCC. Rockville. 51 pp. Booth, C. 1970. Fusariut oxysporut. CHI Descriptions of Pathogenic Fungi and Bacteria.

No. 211. COIIOnN. Hycol. Inst., KeN. Subraaanian, C. V. 1970. Fusariua oxysporut f. sp. conglutinans. CHI Descriptions of

Pathogenic Fungi and Bacteria. No. 213. Coaaonw. Hycol. Inst., Kew. Tuite, J. 1969. Plant Pathological Hethods. Burgess, Hinneapolis. 239 pp. Walker, J. C. 1968. Plant pathology. 3rd ed. HcGraw~Hill, New York. 819 pp.

6

CRUCIFER GENETICS COOPERAT~VE. DEPT. OF PLANT PATHOLOGY, 1630 LINDEN DR., UNIVERSITY OF WISCONSIN, HAOISON, WISCONSIN 53706 (608-262-8638)

DSRS 12-12-86 WILPAU

RHIZOCTONIA DISEASES

Rbizoctonia solani Kuhn (RS)

Tbanstepborus cucu•eris (Frank) Donk

HOSTS Cruciferae and various other vegetables, 250 sp. (Mordue, 1974; Parmeter, 1970).

DISEASE Soil borne, early infection gives seed decay pre & post emergence damping off; later infection on stem gives cankers, wire stem; on older plants, cabbages, a bottom rot of lower leaves, headrot; on root crops, radish, a scurf, turnips etc., canker, root rot; world-wide distribution.

DISEASE CYCLE

1° inoculum sclerotia; ~ mycelium in soil

sclerotia formation on necrotic tissues and soil

colonization of succulent stems, petioles mature head, leaves, rots

HOST PROPAGATION

direct penetration of seed and

seedling parts, damping off

f infection of

lower stem cortex, cankers,

wirestem

Seed sown 0.5 em deep singly in individual pots of 12-pack multipot in •peatlite', 24C, 18-24 hr, 250 ~moles-1m-2 for 14 days.

1

.,..,.,_, ... lc

. tf·-' .. , J

TARGET TISSUE Hypocotyls of 14-day-old and older seedlings.

INOCULATION With your fing.ers, deposit a single inoculum propagule (RS-infested maize kernel) 1 em away from the base of each young plant (Howard and Williams, 1976). With a thin rod, press the propagule into the soil to a depth of 0.5 em and lightly cover with soil.

INCUBATION

, ... c.,.,J"'­P'•P•1-'• ~-~ ··i~ .. "'-.........

Infectional environment; no special environment needed, grow at host propagation environment. Incubation environment; same as above. Grow plants 3 weeks or more and observe progression of disease periodically.

EVALUATION Observe interaction phenotype (IP) development periodically from 5 days post inoculation (PI) and record final IP's at 3 weeks PI or later. Interaction phenotype: symptoms will range from a rapid colonization of hypocotyl accompanied by girdling of stem and damping off, to development of spreading to more restricted lesion at and below the soil line; resistant types have more restricted lesion development. Development of IP's depends very much on the degree of succulence of the seedlings at the time of inoculation. Younger more succulent tissue is more suspectible. Different scales should be prepared for plants inoculated at later times; at time of final rating plants should be pulled, washed and observed at the hypocotyl region for symptoms.

2

Scale; plants scored 0-9 at 14 days or more. O=no darkening of hypocotyl, plants as in

uninoculated controls. l=small, 1-few mm, dark superficial lesions on

hypocotyl at or below soil level, normal plant growth.

3=more extensive limited superficial lesions often acurfy, at or below soil level lesions primarily at 2 root exit points

5=larger, 5 mm-1 em, lesions, superficial very slowly enlarging, restricted distinct margins, sometimes girdling but always superficial, no stunting of plant growth.

7=extensive, enlarging lesions below and above soil level, lesions not girdling but deeper than in superficial surface tissue, plant may be stunted or not.

9=plants girdled by rapidly expanding lesions, necrosis, death.

0

DISEASE INDEX 9

DI = 2:_ {i)(j) i=o n

n=total plants, i=IP class, j=no. plants/class

SELECTION susceptible controls, IP=7-9 by 7 days PI; partial resistance, IP=3-5; resistant plants, IP=0-1.

REFERENCES

Grisham, M.T. 1978. "Variations in the pathogenicity and host specificity of isolates of Rhizoctonia solani associated with carrots." Ph.D. Thesis, University of Minnesota, St. Paul. 67p.

Howard, R.J., and P.H. Williams. 1976. Methods for detecting resistance to Pythium and Rhizoctonia root diseases in seedling carrots. Plant Dis. Reptr. 60:151-156.

Mordue, J.E.M. 1974. Thanatephorus cucumeris. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 406. Commonw Mycol. Inst., Kew.

Parmeter, J.R., Jr. Ed., 1970. Rhizoctonia solani, Biology and Pathology. Univ. Calif. Press, Berkley. 255 pp.

3