Bulletin 764 (tehnica
Bulletin 764 (technical)
BIOLOGY, MORPHOLOGY, TAXONOMY
AND DISEASE RELATIONS OF
THE FUNGUS BREMIA
R. B. Marlatt
Agricultural Experiment Stations
Institute of Food and Agricultural Sciences
J. W. Sites, Dean for Research
University of Florida, Gainesville
BIOLOGY, MORPHOLOGY, TAXONOMY
AND DISEASE RELATIONS OF
THE FUNGUS BREMIA
Robert B. Marlatt
University of Florida
Agricultural Research and Education Center
This public document was promulgated at an annual cost of
$649.99' or a cost of 93 c per copy to provide information on
the fungus genus Bremia, which causes downy mildew of lettuce.
I. Introduction.......................................................................... 1
II. Distribution ..................................................... ................ 1
III. Hosts......................................................... ................... 2
Inoculation ....................................................................... 5
Infection ....................................................................... 6
Colonization ..............................................:..................... 7
Sporulation ..................................................... .............. 8
Symptom s ......................................................... ..... 10
Overseasoning ................................................ ............ 12
V. The FunSus
Taxonomy ......................................... ................. .............. 13
Conidiophore ................................................. ............ 14
Conidium ......................................................................... 14
Germ tube ....................................................................... 16
Infection hypha.................................................. ........... 16
Vesicles.................................................. .................. 17
Haustorium ......................................... ................... 17
M ycelium .................................................... ............... 18
VI. M icrotechnic.............................................. ........................ 18
VII. Bibliography .................................................... .............. 20
The information contained herein is the result of literature review
and the author's research, which for several years was devoted pri-
marily to downy mildew disease of lettuce (Lactuca sativa L.). This
is a study of a fungus genus, Bremia, which causes the serious
mildew. The disease appears to exist wherever this crop of interna-
tional importance is grown. The study was motivated by a conviction
that better knowledge about the fungus and the diseases caused by
it will lead to more effective disease prevention and control. It is
also hoped that this monograph can serve as supplementary reading
material for plant pathology students.
Lettuce downy mildew was first reported in the United States near
Boston in 1875 (89). Bremia lactucae Regel was reported to be a
pathogen of many composites in Europe and America as early as
1843 (5, 6, 71). In 1921, there were serious losses from downy
mildew in lettuce grown in greenhouses in Iowa (43). More than 80%
of the lettuce crop in the Lower Rio Grande Valley of Texas was
destroyed in 1958-59 (81). The winter crop in the Izmer and Ankara
regions of Turkey was also damaged periodically (9).
Modern methods of trimming and packing head lettuce in the
field, quick vacuum cooling, and reliable refrigeration in transit have
prevented serious deterioration by mildew. If these methods are not
followed, downy mildew can spread in transit and storage from
wrapper leaves to outer head leaves, causing brown discoloration of
the latter (57).
Before facilities for quick refrigeration were available, secondary
rots also frequently occurred. Alternaria sp., Botrytis cinerea Pers.
ex Fr., and bacteria occasionally destroyed shipments before they
reached market or at the terminal (35, 37, 44). Artichokes (Cynara
scolymus L.) bearing lesions caused by B. lactucae have been
further damaged by secondary invasions of B. cinerea and Asochyta
cynarae Maffei when the produce is stored in polyethylene bags (47).
Bremia lactucae Regel has been reported in the following areas:
Algeria; Argentina: Tucuman; Australia: Northern Territory, Queens-
land, South Australia; Belgium (14); Brazil (2): Bahia, Rio Grande
de Sul; Bulgaria; Canada: British Columbia, Manitoba, Nova Scotia,
Ontario, Prince Edward Island, Quebec, Saskatchewan; Chile (14);
China (34, 46, 53, 82); Congo; Cyprus; Czechoslovakia; Denmark;
Dominican Republic (14); England (22, 50); Ethiopia (14); Formosa
(23, 79); France (86); Germany (14); Hungary (60); Iceland (14);
India (54); Indonesia; Ireland (14); Israel (58); Italy (14); Japan (23,
82); Kenya; Latvia; Lebanon; Morocco; Netherlands; New Zealand;
Northern Ireland (14); Norway (29); Pakistan (West); Panama; Peru;
Philippines; Portugal; Rumania (14); Santa Domingo (12); Sardinia;
Scotland (14); Spain (36); Sweden; Switzerland (14); Tunisia (32);
Turkey (9); U.S.S.R. (16, 82): Kirizia (14), Siberia (48), Turkman
(14), Ukraine (45); U.S.A. (76): Arizona (39), California, Colorado
(83). Florida (83. 88). Idaho (72). Iowa. Maryland. Massachusetts.
Minnesota, Mississippi, Missouri (83), Montana (72), New York (83),
North Carolina (14), Oklahoma (83), Oregon (72, 73), Pennsylvania,
Texas, South Carolina (83), Washington (72, 73, 83), Wisconsin (83);
Venezuela (14); Yugoslavia (42); Zambia (14).
Other species of Bremia have been reported in the following areas:
B. centaureae Syd.: Israel (58), Norway (29); B. domingensis Cif.:
Dominican Republic (12); B. elliptica Saw.: China (46), Japan, For-
mosa (23); B. graminicola Naum.: China (46), India (54), Japan (82),
U.S.S.R.: Siberia (48); B. graminicola var. indica Patel: India (54);
B. lactucae f. chinensis Ling & Tai: China (46); B. lactucae f.
souchicola (Schlecht.) Ling & Tai: China (46); B. lactucae f. taraxaci
(Ito & Tokunaga) Ling & Tai: China (46); B. lampsanae Syd.:
France (86), Norway, (29); B. microspora Saw.: Formosa (79); B.
ovata Saw.: Formosa (79); B. picridis Ito & Tokunaga: Japan (82);
B. 'ausureae Saw.- China (34). Formosa (79): R. sonchi Saw.:
Formosa (79), Norway (29), Rumania; B. sonchicola (Schlecht.) Saw.:
China (53), Norway (29); B. taraxaci Ito & Tokunaga: Japan (23),
Norway (29); B. tulasnei (Hoffm.) Syd.: England (22), France (86),
The preceding list points out that distribution of Bremia is
worldwide, occurring on all continents but Antarctica. This wide
distribution greatly contributes to the importance of diseases caused by
the fungus. Not only is lettuce damaged, but, on the positive side,
a great many weeds become diseased.
Hosts of Bremia sp. are as follows: Bremia centauriae Syd.:
Centauria cyanus L., C. ferox Desf., C. hyalolepis Boiss. (58), C
jacae L.. C iacae augustifolia, C macrocephala Puschk.. C. melitensis
L., C. montana Auct. (77), C nervosa Reichb., C. nigricana DC.,
C phrygia L., C rapontica, C. rutifolia Boiss., C. sp. (29); B.
domingensis Cif.: Parthenii hysterophori L. (12); B. elliptica Saw.:
Lactuca formosanae Maxim., L. lacinata Makino, L. raddeanae Max-
im. (23); B. graminicola Naum. var. indica Patel: Arthraxon ciliaris
Beauv. (46, 48, 53, 82), A. lancifolius Hoch (54); B. lactucae:
Agoseris glauca (Pursh.) Raf., A. grandiflora (Nutt.) Greene, A.
heterophylla (Nutt.) Greene (72, 73), Andropogon dandelion (64),
Arctium lappa L., Carduus acanthoides L., C arvensis (50), C.
crispus L. (29), C defloratus L. (64), C. pycnocephalus L. (9),
Carthamus tenuis Boiss. (58), C. tinctorius L., Centauria cyanis L.
(29, 56, 64, 72, 73, 83, 89), C. ferox Desf. (64), C. imperialis Hort.
(7), C. jacea L. (29, 56, 64), C. jacea var. augustifolia, C. macro-
cephala Puschk, C. melitensis L., C. montana L., C. nervosa Reichb.
(64), C. nigra L. (7, 29, 64), C. nigrescens DC, C. nigricans DC.,
C phrygia L., C rapontica, C. rutifolia Sibth. & Sm., C. uniflora L.
(64), Cichorium endivia L. (3, 56, 64, 88, 89), C. intybus L. (64,
83), Cineraria hybrida Bernh. (64), Cirsium anglicum DC., Cirsium
arvense (L.) Scop. (3, 29, 56, 64, 86), C. bulbosum DC. (64), C.
boujarti Pill. & Miller, C. canum All. (56, 64), C. carniolicum Scop.,
C. ciliatum Moench. C. erisithales Scop. (64). C. lanceolatum Hill.,
C. oleraceum Scop. (56, 64), C. palustre Coss., C. pannonicum Link,
C. pauciflorum Koch, C. pyrenaicum DC. (64), C. vulgare (Savi)
Airy-Shaw (29), Crepis aspera L. (59), C. aurea Reichb. (64), C.
biennis L. (56, 64). C. blattarioedes Vill. (64). C. hulhosa (I..)
Tausch. (58, 64), C. foetida L. (64), C. grandiflora Tausch. (56, 64),
C. mollis Aschers (64), C. paludosa (L.) Moench. (29, 56. 64),
C. rubra L., C. sibirica Gouan (64). C. tectorum L. (29. 56. 64). C
vesicaria L. (64), C. vesicaria taraxicifolia (64), C. virens L. (56,
64), Cynara cardunculus L. (56, 64, 86), C. scolymus L. (32, 84),
Dendroseris macrophylla D. Don, Dimorphotheca aurantiaca D. C.
(64), D. pluvialis Moench. (86), Erechtites hieracifolia Rafin (83),
Gaillardia aristata Pursch (22), G. grandiflora (7), G. picta Sweet
(86), G. pulchella Faug. (7). Gnaphalium sp. L.. Helichrvsum hrac-
teactum Andr. (29, 42, 86), H. chrysanthemum (56, 64), H. macran-
thum Benth. (56), Hieracium amplexicaulis L. (64), H. aurtiacum
L. (64, 83), H. auricula L. (64), H. borealis Fr. (56, 64), H. laevigatum
Froel., H. lanatum Brot. (64), H. murorum L. (56, 64), H. picroides
Vill. (64), H. pilosella L. (56, 64, 86), H. praealtum Vill., H. praecox
Tausch (64), H. pratensis Tausch (56, 64), H. prenanthiodes Doell,
H. pulmonarioides Vill., H. riparium Juxip. H. rubrum Peter. H. sa-
baudum L. (64), H. stoloniflorum Thuem. (56, 64), H. umbellatum L.
(29, 56, 64), H. villosum L., H. viscosum Arv. (64), H. vulgatum
Fries. (56, 64), Hypochoeris glabra L. (29, 56, 64), H. radicata L.
(29, 56, 64), Jacobaea sp. (64), Krigia biflora (Walt) Blake (83),
K. dandelion Nutt. (76), Lactuca alpina (L.) Gray (29). L. altissima
Bieb. (3, 6, 56, 64), L. augustana (3, 89), L. biennis (72), L. cana-
densis L. (6, 18, 43, 64, 76, 83), L. chinensis F. N. (34), L. crispa Roth
(6, 56, 64), L. elongata All. (3), L. hirsuta Muhl. (64), L. indica L.
(34), L. integrifolia Bigel (6, 76), L. japonica Regel (3. 56), L.
lesicophaeae (6), L. leucophoea Gray (76), L. ludoviciana (Nutt.)
Riddell (18, 43, 64, 72), L. muralis E. Mey. (6, 55, 64), L. palmata
Willd. (3, 56), L. perennis L. (64), L. pseudovirosa (3), L. pulchella
(Pursh) D. C. (64, 72, 73, 83), L. sagittata Hook. (6, 56, 64),
L. sagittifolia Ell. (18, 43, 64), L. saligna (93), L. sativa L. (2,
3, 56), L. sativa 'Asparagina' (51, 73), L. scariola L. (6, 9, 18, 64,
93), L. scariola var. integrate (43), L. serriola L. (51, 55, 56, 72,
83). L. sibirica (L.) Benth. (29), L spicata (Lam.) Hitchc. (64. 73,
83), L. viminea J. & C. (64), L. virosa L. (36, 51), Lagoseris bifida
(Vis.) F. & M. (58), Lampsana communis L. (3, 29, 56, 64), L.
virosa L. (64). Lappa major Gaertn. (56. 64), L. minor Hill, L.
officinalis All. (64), Leontodon autumnalis L. (29, 56, 64) L
hispidus L. (56, 64), L. hispidus hastatus (64), Melichrysum bractea-
tum, Mulgedium alpinum (L.) Less. (56, 64), Nabalus albus Hook.,
N. altissimus Hook (64), Parthenii hysterophori L., Picris galilea
(Boiss.) Benth. & Hook. (58), P. hieracioides L. (86), Pieris hiera-
cioidis L. (56, 64), Prenanthes alba L. (83), Rhodanthe manglesii
Lindl., Senecio abrotanifolius L., S. alpinus L. (64), Senecio altiacus
Sch. Bip. (17, 68), S. aquaticus Hill., S. arvensis Rouy, S. asper Ait.,
S. cracaefolius Willk. (64), S. cruentus L. (2, 64), S. elegans L.
(7, 64), S. hybridus Hort. (64), S. jacobeae L. (55, 56, 64, 93),
S. jaquemontianus Rchb. (29), S. oleraceus Crev. & Lem., S. rupester
(64),. S. squalidus (55), S. sylvaticus (55), S. vernalis Hoppe (56,
64), S. viscosis L. (17, 55), S. vulgaris L. (3, 17, 21, 22, 29, 50,
55, 56. 64, 93), Solidago virgaurea L. (29), Sonchus arvensis L. (3,
17, 29, 56, 64, 83), S. asper (L.) Hill (16, 29, 50, 56, 64, 72, 73, 83),
S. fallaciis, S. fallax Walb. (64), S. longifolius Trev., S. oleraceus
L. (21, 29, 50, 56, 64, 83), S. palustris, S. tingitanus Lam. (64),
Taraxacum ceratophori D. C., T. mongolicum Hand., T. officinalis
Knaut. (17, 64), T. platycarpum Dahlst., Taraxacum vulgaris Schrenk.,
Tragopogon major Jacq., T. minor Fries (21), T. orientalis L. (64),
T. pratensis L. (56, 64), T. sp. (29); B. lactucae f carthami Mil:
Carthamus tinctorius; B. lactucae Regel f. chinesis Ling & Tai:
Lactuca chinensis Makino; B. lactucae Regel f. ovata (Saw.) Ling &
Tai: Crepis japonicus Benth.; B. lactucae Regel f. sonchicola
(Schlecht.) Ling & Tai: Sonchus oleraceus L.; B. lactucae Regel f.
taraxaci (Ito & Tokunaga) Ling & Tai: Taraxacum mongolicum
Hand. (34); B. lampsanae Syd.: Lampsana communis L. (29, 78, 86);
B. microspora Saw.; Lactuca debilis Mass., L. dentatae 'Thunbergii'
Makino (23); B. ovata Saw.: Crepis japonica Saw. (79); B. picridis
Ito & Tokunaga: Picridis hieracioides L. var. japonicae Regel (23);
B. saussureae Saw.: Henrisepta carthamoides Kuntze (24, 79), Saus-
surea affinis (34); B. sonchi Saw.: Sonchus oleraceis L. (79). S.
palustris L.; B. sonchicola (Schlecht.) Saw.: Sonchus arvensis L.
var. uliginosus Travtv. (53), S. asper (L.) Hill, S. sp. (29); B.
taraxaci Ito & Tokunaga: Taraxacum ceratophori D. C., T. platycarpi
Dahlat. (23), T. sp. (29), T. vulgaris Schrenk. (23); B. tulasnei
(Hoffm.) Syd.: Senecio jacobaea var. vulgaris L. (86), S. vulgaris
L. (29, 77).
The customary treatment given lettuce downy mildew in plant
pathology texts rarely touches upon the tremendous host range of
the fungus. It is enlightening to learn that at least 230 different
species are susceptible to the fungus and there may be more discovered.
Combined with its worldwide distribution, this extensive host range
adds to the importance of Bremia.
Several methods of inoculation have been devised, all involving
application of conidia to host foliage. For example, infection occurred
when a water suspension of conidia was sprayed onto the soil surface
shortly before lettuce seedlings emerged (50). As the seedlings broke
through the soil surface, they became coated with conidia. Leaves
have also become infected when their surfaces were lightly abraded
with celite before being inoculated with a conidial suspension. In
one test this abrading lowered the mildew resistance of one lettuce
variety (11). For this reason, such inoculation cannot be recommended
for tests for resistance.
Lettuce has been inoculated in the laboratory in Petri dishes
(59). The inoculum was prepared by suspending conidia from five
diseased cotyledons in 25 ml of water for spraying. Seeds were sown
onto moist filter paper in the dishes at the rate of 200 to 300 seeds
per 12 cm dish. They were stored at 100C for 3 days in either light
or darkness. On the fourth day the dishes were stored at 15C
in light. Within 8 days cotyledons emerged from the seeds and were
inoculated daily with conidial suspensions for as many as 14 days.
If inoculated only once, 2 to 3 days were required to reach 100%
infection (59). Leaf discs were used in one instance instead of seed-
lings, but inoculation was less successful (28).
The most common method of inoculation involved growing seed-
lings in flats or pots. Plants that furnished the inoculum were grown
in a saturated atmosphere until covered by conidiophores and conidia.
The plants' foliage was then shaken in distilled or fresh tap water,
and the resulting conidial suspension was filtered through cheese-
cloth and sprayed onto the new hosts with an atomizer (38, 39, 50).
The incubation period was shortest if a thin film of water covered
the entire leaf. Flats of seedlings were subjected to repeated inocula-
tions with roguing of infected plants In this manner only resistant
plants remained and were thus selected (59, 26). Inoculation was
most successful if cotyledons were inoculated as soon as they un-
folded and inoculum was applied to upper and lower surfaces (59).
The two to three-leaf stage was also readily inoculated (39). When
races of the fungus were being investigated, single-spore cultures
were sometimes used (10, 11).
The optimum environment for conidial germination and infection
was 45 hours of darkness, including 24 hours of 100% relative humidity
at 6 to 100C, followed by 7 to 10 days under moderate humidity
and 480 to 500 foot-candles of light (50, 55). Later plants were held
again for 1 or 2 days in 100% relative humidity to induce sporulation
(55). If the 7 to 10-day period was omitted, conidia failed to develop
Infection consists of the fungus entering the host and continuing
to grow while utilizing nutrients provided by the host's cells. In one
instance, zoospores were released from sporangia and penetrated host
stomata (44). Such zoospores were reported to be attracted by
hydroxyl ions (2). However, zoospores are rarely observed; conidia
usually germinate directly, and a hypha penetrates the host (2, 62).
Conidia begin to germinate 1 hour after inoculation. After 3
hours, one-third had germinated on lettuce cotyledons, and all had
germinated 6 hours after inoculation. Germination was slightly slower
on agar (39).
In most instances conidia formed infection hyphae which penetrat-
ed epidermal cells 3 hours after inoculation (39, 55). Stomatal pene-
tration was rarely seen, even though germ tubes actually passed
over open stomata. However, a few strains of the fungus penetrated
stomata (13). Generally the germ tube penetrated the cell wall with
the aid of an appressorium. Appressoria were said to have formed
in vitro, often very close to the conidia (85). After 10 to 12 hours most
infections had occurred.
The time required for epidermal penetration was determined in
one experiment by fixing and staining at hourly intervals or by killing
the fungus with zineb at intervals and noting whether or not disease
resulted. Infection was prevented by spraying with zineb within 3
hours at 100 to 220C or within 6 to 8 hours at 40 to 80C (85).
Five to seven hours of high relative humidity are required for
infection. This is one of the reasons why lettuce downy mildew
incidence in California increased toward the more humid Pacific coast
and decreased toward the drier mainland. Since the optimum tempera-
ture for infection was 150 to 170 C, disease incidence was also lowered
toward the warmer inland areas (61). Temperatures of 10 to 200C
in a saturated atmosphere were conducive to infection, but at 250
C no infection took place (55).
Ninety per cent of the small infection hyphae penetrated anti-
clinal wall boundaries of the epidermis and 50 the periclinal walls.
Following anticlinal wall penetration, the hyphae then entered the cell
lumen from an area slightly below the surface of the periclinal wall
(2). In one experiment germ tubes were attracted to the cuticle of
Lactuca sativa and Senecio cruentis and penetrated the epidermis of
both, but only L. sativa was colonized (2). It was suggested that the
tubes were attracted to anticlinal walls by accumulated alkaline mate-
rials at those locations. The number of leaves that became infected
was correlated with total sugars and reducing sugars in the leaves
Fewer conidia produced at 200 and 22 C germinated than those
produced at 100 to 15C. Conidia that were hyaline and minutely
granular were viable; those which were nearly opaque and coarsely
granular did not germinate (85). Conidia were viable on glass for a
maximum of 7 days in high humidity; however, most died on dry glass
within 4 days.
In addition to leaves, the outer cortical parenchyma of flower
stalks became infected, as well as stalk leaves (85); however, no in-
fection could be obtained in the capitula. Roots and hypocotyls
were inoculated with a conidial suspension, and only the hypocotyls
became infected (41). Retarded plants are often disease prone. Re-
tarded lettuce, however, was no more susceptible to mildew infection
than vigorous plants.
Before 1962 the lettuce downy mildew fungus was considered to be
confined to the parenchyma of the leaf lamina, the parenchyma of the
adjoining petiole, and leaves and cortex of the flower stalk. The
hyphae rarely invaded parenchyma around the largest leaf veins and
the center of the petiole (85). One strain of Bremia lactucae was
found in the cortex of stems of Senescio vulgaris.
In 1962, systemic colonization of domestic lettuce was established
(39). Intercellular hyphae were found 54 hours after inoculation of
seedling cotyledons and leaves. After 66 hours hyphae averaged 141
us in length and a few had begun to branch; the average length was
309 i after 72 hours, and hyphae grew in all directions between
leaf mesophyll cells.
Hyphae in the base of cotyledons (but rarely from leaves) grew
into hypocotyls 90 hours after inoculation; the hyphae averaged 2.2
mm at that stage. Most of them entered the hypocotyl via tissue
immediately under the epidermis; a few grew adjacent to xylem and
along the sides of laticifers. Some hyphae grew from one cotyledon
into the other, and additional hyphae entered the first leaf from the
cotyledons after 108 hours.
Occasionally some hyphae grew from the cotyledon to the hypo-
cotyl and down the tap root after 114 hours, but most often root
invasion required a minimum of 150 hours. Hyphae grew down the
tap root in tissue immediately under the epidermis or that adjoin-
ing the stele. After 156 hours some branch roots were invaded, and
by this time hyphal growth down the root averaged 2 mm. Lateral
roots were colonized by hyphae close to the epidermis; those near
the stele grew down the tip of the tap root 188 hours following
inoculation of cotyledons (39). Mycelium in the outer cortical paren-
chyma of flower stalks spread 5 to 10 mm during 10 weeks (85).
The main hyphae in a leaf were described as dichotomous, but
often they filled the intercellular spaces of mesophyll, which resulted
in a variety of branching (3). Optimum conditions for hyphal growth
within a lettuce leaf are high relative humidity of outside air and
200 to 220 C (1). This temperature is the highest optimum reported.
Other studies showed that the fungus in lettuce proceeds to spread
at 90 to 180 C (optimum 120 150 C). Disease was sometimes
arrested at less than 40 and higher than 180 C (67). Once arrested,
the disease started again when lettuce was kept for a while at 40 C.
If held "too long" at 240C, the disease was permanently stopped.
The fungus died in leaves kept at 320 C for 48 to 72 hours (67).
The lettuce downy mildew incubation period has been divided into
three phases (70). Phase I includes the first 3 days after inoculation,
during which darkness stimulates disease development. During the next
3 days, Phase II, darkness somewhat retards disease. Phase III includes
days 7, 8, and 9, during which darkness seriously retards the progress
of the disease. Darkness includes wave lengths of 350 to 500 mu.
Ultra violet light inhibits disease, possibly due to falling glycolytic
activity. During stage III, as the fungus prepares for sporulation, low
light may retard disease because of decreased respiration. High temp-
eratures are tolerated well during Phase I and II, but cause re-
gression of disease during Phase III.
Colonization of lettuce is inhibited if oxygen in air rises over 80%.
More than 12% carbon dioxide also inhibits colonization.
During the first 4 weeks of disease in young plants systemic
infection proceeded rapidly, accompanied by the appearance of ab-
normally large, thick-walled haustoria. During the 5th and 6th week
most hyphae in roots were distorted and surrounded by dead host
tissue. Fragments of normal hyphae were infrequently found. Seven to
10 weeks after inoculation all root hyphae were distorted and broken
In hosts other than lettuce, mycelium and haustoria of B. lactucae
were found in the stem pith of Sonchus oleraceous and in the stem
cortex of Senecio vulgaris (21).
After the fungus has colonized lettuce, hyphae accumulate under
leaf stomata in preparation for sporulation. Incubation periods (inocu-
lation to sporulation) varied from 5 to 8 days, 6 to 8 days, 9 to 12
days, and 3 to 4 weeks (55, 65). The length of incubation period
diminished as the number of conidia in the inoculum increased (11).
For example, 1000, 10,000 and 100,000 conidia gave 0, 27%, and 67%
sporulation in 7 days and 30%, 82%, and 99% sporulation in 9 days,
respectively. The higher the concentration of inoculum, the sooner
In one experiment inoculated young plants were kept in 100%
relative humidity; conidiophores were produced 162 hours after inocu-
lation, and sporulation occurred after 168 hours (39). Before sporula-
tion occurred, the mycelium had to fill many substomatal cavities
(85). Sporulation began near the point of inoculation. The conidio-
phore vesicle was larger, and had more sterigmata, and more conidio-
phores arose from stomata when humidity was high (64).
Without a film of water on a leal, no conidiophores grew (85).
If there was just one drop of water on a leaf, conidiophores arose
in a narrow ring around the drop. With no water film, infected areas
turned yellow and died, with no sporulation. If moistened later, sporu-
lation occurred at the margin of the dead spot. This requirement for a
water film explains the numerous reports that high relative humidity
is required for sporulation (85). Relative humidity had to be lowered
after inoculation and later raised again for maximum sporulation. I his
must depend on other circumstances, however, because another report
recommended continuous 100% RH to produce conidiophores 162
hours after inoculation and conidia after 168 hours (64). The optimum
vapor pressure deficit for sporulation was 0 mm, with a range of
0 to 1.8 mm (61).
The cardinal temperatures for sporulation have reportedly been
40 or 50 C minimum, 240 C maximum, and an optimum of 100
to 210 C (55). After a proper incubation period under optimum
conditions, sporulation has occurred as early as the end of the 5th
day (39). In this particular instance temperatures ranged from 200
to 22C (
At the end of the incubation period the lettuce mildew fungus
did not first sporulate in daylight; sporulation took place 7 hours after
the following night began. Before application of a water film to leaves,
a dark period hastened sporulation. However, if that dark period was
preceded by 1-A hours of light, no sporulation took place until the
next night (65). The light period consisted of 10,000 lux of light
at 150 to 200 C (85).
In another experiment, downy mildew infected lettuce leaves were
kept moist and dark at 220 C and all of 12 infected spots sporulated
(94). Under the same conditions, exposure to 800 foot-candles of
light permitted only I of 13 infection spots to sporulate (94). In
another test, lettuce was inoculated and kept in the dark for 7 to 9
days, and sporulation was sparse. Another group of plants received
light from the time of inoculation, and heavy sporulation occurred
on the 7th to 9th day. When a third group was kept dark for 6 days
after inoculation then given light, that following night sporulation
was profuse. Some of these reports conflict, indicating that factors
other than light are influential.
Sporulation adversely affects lettuce leaves. Young leaves or
cotyledons were green when conidia were first produced but began to
yellow after a few more days at 50 to 150 C. At higher temperatures,
leaves completely gelatinized after 2 to 3 days of sporulation (if
most of the leaf was infected). With spotty infection, the spots gradually
increased in size and their centers became necrotic. If leaves were
entirely covered with sporulation and were allowed to dry, they died
within I or 2 days (85). Mildew alone does not kill lettuce; it is the
combination with secondary pathogens.
It is obvious that reported incubation periods and optimum condi-
tions for sporulation vary widely and often conflict. This is under-
standable, since incubation is slowed by ultra-violet light, darkness
during later periods of incubation, high oxygen or high carbon dioxide,
low relative humidity, and low amounts of inoculum. Even the amount
of water on a leaf can change these periods.
Head, leaf, and cos lettuce were more susceptible in the early
stages of their growth than they were as maturity approached
(63). Some plants were inoculated when they were 21, 31, and 45
days old. The percentages of mildew that resulted were 90%, 38%,
and 19% respectively (18). Cotyledons were especially susceptible,
and sporulation occurred on both under and upper sides (74). Leaves
4 cm or more in length were less susceptible. For this reason downy
mildew often was most rampant in seed beds or in rows before
thinning. The crowded, moist environment of a seed bed was also
conducive to epiphytotics (43).
Symptoms on moderately infected young leaves and on older
leaves consisted of yellowish-green spots sharply delimited by any
veins which had more than three spiral vessels (62). As the disease
progressed, only larger veins obstructed the spread of lesions, but
the spots remained angular in outline due to veinal obstruction. Much
more sporulation was seen on the undersides of leaves than on upper-
sides (74). Symptoms on mature leaves of susceptible varieties, i.e.,
scattered lesions, resemble symptoms on young leaves of varieties
with some resistance.
If growing conditions caused leaves to have small parenchyma
cells and narrow intercellular spaces, lesions were glassy instead of
yellow, separation of infected areas by veins were more marked,
and leaves eventually became necrotic. Another variation in symptoms
resulted from keeping plants in the dark for 7 to 9 days after inocu-
lation. The leaves became flaccid, rolled, and eventually necrotic
(65, 70). If plants were kept dark for only 6 days and then given
light or were given continuous light, the usual symptoms were ex-
pressed. Downy mildew severity has been graded in five categories:
1. one to five -conidiophores per leaf as observed microscopically,
2. some conidiophores readily evident to the naked eye, 3. a thin coat-
ing of conidiophores evident to the eye, .4. leaf fairly severely damaged,
5. entire leaf very severely damaged.
Systemically infected head lettuce at its marketable stage had in-
ternally cracked stems with darkened pith and oozed latex excessively
when cut. Leaves on seedstalks occasionally were diseased and
showed typical symptoms.
When seeds were inoculated just before germination, the emerg-
ing root was discolored brown (59). This symptom was found on both
resistant and susceptible varieties. The production and quality of
seed was substantially reduced when seed plants had downy mildew
A lettuce crop with a high incidence of infection matured 6 days
later than a similar healthy crop (93). When three leaves of lettuce
were infected in the three to four-leaf stage, plants yielded lighter
heads of lower quality than lettuce with only one or two leaves in-
fected while young (74).
One study revealed that mildewed lettuce leaves had an average
temperature of 1.590 C above the surrounding air, whereas healthy
leaves averaged 0.630 C higher (95). Because of this difference of
approximately IC, this disease should lend itself to detection by
Studies of the physiology of infected leaves revealed that the
glycolysis respiration ratio was 0.32/0.70 as compared to 1/1 of
normal leaves (69). In another case, respiration was increased, but
no change in glycolysis was detected (66). By studying infected leaf
pieces, it was noted that the fungus made use of glucose, lactose,
and saccharose more than levulose and galactose; xylose was used least
Other lettuce pathogens may or may not be associated with downy
mildew. For instance, powdery mildew (Erysiphe cichoracearum D.
C.) has been seen to grow over downy mildew lesions on lettuce
(61). Downy mildew lesions may also serve as portals for entry of
secondary pathogens (74, 92, 96). Botrytis cinerea readily invades
mildewed lettuce, and a storage test showed that at 50 C more bacteria
and fungi rotted mildewed lettuce than healthy lettuce (93). Downy
mildew has been found to make lettuce plants more susceptible to cold
injury in the field (74).
The occurrence of B. lactucae strains on hosts other than domestic
lettuce has been frequently reported, but descriptions of symptoms
on such hosts are limited. Lactuca serriola infected by Bremia lactucae
had lesions somewhat like those on domestic lettuce, except they were
smaller and more widely scattered, and infection was generally
more difficult to obtain. Bremia graminicola infection of Arthraxon
ciliaris showed pale yellow lesions; they either were delimited by
veins or had coalesced to include the entire leaf (46, 48, 82). The
leaf underside was covered by grayish conidiophores. Later, severely
infected leaves wilted, shriveled, and dried to a brown color.
B. graminicola var. indica on Arthraxon lancifolius formed whitish
to yellow-brown lesions. The undersides of the spots were wooly with
whitish, later grayish conidiophores (54). Foliage of Gaillardis and
Helichrysum sp. was nearly destroyed by downy mildew; stems and
peduncles dried, and seed did not form (86).
Wild lettuce in Iowa was suspected as a means for overwintering
of B. lactucae (19). Suspected plants included Lactuca canadensis, L.
ludoviciana, L sagittifolia and L scariola var. integrate (43). Similarly,
in England two biennial weeds, L. serriola and L. virosa, were report-
ed to carry the fungus between lettuce crops (52). Although L
serriola and volunteer L. sativa were able to live in shade through
the summer in the high temperatures of the Arizona low-altitude
desert, experiments revealed that the fungus did not remain viable
within these hosts throughout the hot season (41). However, during
summers near Chentu, China, shaded volunteer lettuce plants were
found with downy mildew (34).
Lettuce debris in soil was found to harbor the fungus' mycelium
near Chentu, China, and in Iowa (19). It has also survived from
one year to another in hothouse soil when the soil did not freeze
(19). However, mildew is rarely soil transmitted. For instance, in Great
Britain mildewed leaves were incorporated in soil, and after several
months the soil ivas planted to lettuce. Only one infected seedling in
5,000 was found (93). Additional evidence that lettuce debris rarely
transmits the disease resulted from failure of the pathogen to become
established after inoculation of lettuce seedlings with mildew lesions
that had been dried and macerated at room temperature (41). Occa-
sional downy mildew found on lettuce seedlings grown in petri dishes
suggested a seed-borne disease. The likelihood of B. lactucae being
borne with lettuce seed was found to be only 0.025% in one test (93).
Since, theoretically, only one conidium is sufficient to start an epiphy-
totic, these rare cases of transmission must be considered.
Despite reports that the lettuce disease probably overseasons as
fungus oospores (87), rarely has proof been offered. Oospores were
found in domestic lettuce in Norway in 1964 and in the following
weeds: Lactucae scariola, Lampsana communis, Leontodon autumnalis,
Senecio vulgaris, Sonchus arvensis, and S. asper (29). The only other
reports found referring to oospores in lettuce were accounts of fruit-
less searches for them.
To summarize, disease usually results when air-borne conidia fall
upon a congenial host and environmental conditions are conducive to
infection and colonization. Environment also determines the disease
incidence by its effect upon sporulation.
In 1949 it was pointed out that the correct designation of the
genus was Bremia Regel ex. Schroet., or Bremia Schroet.. The binomial
would be Bremia ganglioniformis (Casp.) Shaw, because Caspary ful-
filled the requirements for valid publication (71). This would follow
the International Code of Botanical Nomenclature at that time. The
Code, as published in 1956 by the International Bureau for Plant
Taxonomy and Nomenclature, (Lanjouw et al.) Chapter V, Section 3,
Article 59, states: "In Ascomycetes and Basidiomycetes with two or
more stages in the life cycle . ., but not in Phycomycetes, the
first legitimate name . . applied to the perfect stage takes
precedence (31)." The imperfect stage of this Phycomycete alone
serves to identify the fungus. Hence Bremia lactucae Regel can stand
Taxonomically the fungus' position is as follows: Phycomycetes,
Oomycetes, Peronosporales, Peronosporaceae, Bremia lactucae Regel,
races 1 through 6. Synonyms: Botrytis ganglioniformis Berk., 1846,
Peronospora ganglioniformis (Berk.) Tulasne, 1854; Peronospora gan-
glioniformis Tul. ex Caspary, 1855; Peronospora gangliformis Berk.
ex de Bary, 1863; Bremia lactucae Regel ex Schroeter, 1886 (20,
56, 60, 87).
The several species of Bremia mentioned under "Distribution"
and "Hosts" were used for the sake of convenience. However, the
author favors the one binomial, Bremia lactucae Regel, because
the great range of morphological features were strongly affected by
moisture and by hosts (64). One possible exception was an interesting
specimen from Tragopogon pratensis which resembled both Plasmo-
para and Bremia (64). As with Plasmopara, the conidiophores' lateral
branches were horizontal, forked two to four times, and unusually
short. However, the tips were drum-shaped swellings with fairly num-
erous sterigmata, as with Bremia. This unusual fungus had very large
conidia with germ pores and sterigma attachments. Before valid
species can be determined, the fungus must be observed over the
entire range of moisture and host differences. Even such a complex,
extensive comparison is unlikely to be valid. The most likely al-
ternative will involve biochemical criteria, since host preference has
been considered incomplete for speciation. Antigenic structure, nutri-
tional requirements, chromatography, electrophoresis, and numerical
taxonomy through the use of computers to assess these relationships
may someday provide the appropriate taxonomic position of the fungus
Races are presntly designated by susceptibility or resistance of
cultivars of Lactuca sativa (90). This system is useful to those who
are interested primarily in lettuce breeding but such races may not be
The prelude to conidiophore development was the formation of
hyphal coils, which often filled substomatal cavities. These coils
bore thickened hyphal tips. Coils which formed 50 to 100 A from a
stoma did not produce conidiophores from the tips (85).
Usually one to three rigid, hyaline conidiophores arose from the
stoma wherever it was located, and were slightly constricted at the
guard cells. At the leaf surface they became circular, 6 to 19 A
in diameter and 200 to 1500 ,A high, gradually tapering toward their
tips. Branching began at one-half to -four-fifths of the distance from
the ultimate top. Branching was dichotomous or rarely trichotomous,
with angles of 900 or more; dichotomous branching occurred three to
seven times. Conidiophores often had septa in their main trunks and
in their branches. It was interesting to note that conidiophore length
was directly related to the relative humidity during growth (64).
At the branch tip a swollen inverted cone or drum-shaped vesicle
formed, 8 to 15 P in diameter. The vesicle bore 2 to 18 sterigmata, the
usual number being 3 to 5. Sterigmata were 3 to 8 p long, 1.5 to 2 A
thick, and rarely branched. They projected from both the inner and
outer walls of the vesicle edge, giving it a stellate appearance. A
sterigma narrowed to a point from which one conidium grew. Only
at maturity, and rarely then, was there any direct relation between
the sizes of sterigmata and conidia (5, 23, 46, 48, 54, 56, 58, 60, 64).
The size of the terminal vesicle and the number of the steng-
mata (and, thus, conidia) diminished as moisture increased. However.
increased moisture caused an increase in number of conidiophores
arising from each stoma. The type of host did not influence the
structure of conidiophores (64).
Conidia were reported to be spherical to elliptical; and lengths
ranged from 12 to 31 A and widths 11 to 27.5 /. depending on
host (5, 23, 46, 48, 54, 56, 58, 64, 85). Volumes of the conidia ranged
from 148 to 389 cu AI. Shape and volume appeared to differ with mois-
ture (64); some conidia became more elliptical in moist air than in
dry air. Referring to conidial size can be misleading, since one cal-
culation revealed that 100 of every 1000 conidial measurements were
erroneous due to conidial age, etc. (64). Between different host
plants, conidial length varied 9.9 A and width 9 A.. Increased humidity
promoted lengthened conidia as much as did host differences, but the
ratios of length to width remained about the same.
One author studied Bremia conidia from many hosts, measured
thousands of them, and separated them into five types (64): 1. small
conidia, between oval and long-elliptical, from Cirsium, Senecio, and
Carduus; 2. medium size conidia, long-elliptical, from Crepis and
Centaurea; 3. conidia medium-large, more or less abruptly elliptical,
from Hieraceum; 4. large conidia elongate to flatly elliptical: on
Lactuca, Lapsana, Pieris and Taraxacum; 5. large, roundish conidia
from Sonchus. Within two groups of Compoiitae, the same worker
found that larger conidia (3 A longer) are produced on members of
the Liguliflorae than on those of the Tubuliflorae (64).
Conidial surface was smooth, the color hyaline. Authors differed
as to whether conidia rarely or usually had one papilla on either
the attachment or the apical end (46, 48, 54, 85). They generally
agreed that the conidial wall was slightly thicker at the papilla.
One report stated that the germ tube grew from the papilla (3).
Conidia remained viable 7 to 10 days it relative humidity was high
(over 50%), and conidia 5 to 6 days old germinated over a wider
temperature range than those 1 day old (80). The older conidia also
tolerated low humidity better than the younger (62); optimum relative
humidity was stated to range from 80% to 100% (55, 61). The use of
sulfuric acid for controlling humidity interfered with germination.
However, the use of zinc sulfate or sodium sulfite was satisfactory
Minimum germination temperature was 30 C. Temperature optima
for germination ranged from 5 100 to 20 250 C, depending upon
the author (34, 43, 55, 62, 85). Conidia produced at 200 and 220 C
had a maximum germination temperature of 31 C and optimum of 4 -
100 C, while those produced at 100 to 150 C had a 290 C maximum
and 2 80 C optimum.
The best substrate for germination of the lettuce strains of
Bremia was found to be lettuce cotyledons (39, 62). In fact, the first
germination took place in less than 1 hour. After 3 hours one-third
had germinated, and all germinated within 6 hours (39). However, on
moist agar only one-fifth of the conidia germinated after 3 hours.
Although it was stated once that nutrients in the substrate did not af-
fect germination (55), others reported certain tap waters containing
calcium and magnesium salts were superior to distilled water (34).
For instance, after 5 days, 62.8% of the conidia germinated in tap
water, while only 23% had germ tubes in distilled water. In lettuce
leaf decoction 85.8% of conidia germinated at 200 C; in tobacco
leaf decoction, 52% (62). Saturated lime water was also reported to
be a good substrate (93).
Conidia were preserved for 6 to 7 months by refrigeration of
sporulating lettuce leaves, but most died within 11 to 12 months
(59). A suspension of conidia in distilled water was frozen to -250 C.;
after storage for 2 months to one year, the suspension was thawed
slowly and conidia were still viable (59).
Conidia rarely developed into sporangia containing biflagellate
zoospores (44). However, in one instance eight or more zoospores
formed per sporangium, when kept at about 100 C in tap water.
At higher temperatures germ tubes were produced from conidia (56).
They were produced over a period of 24 to 48 hours (44).
At 10 to 100 C germ tubes grew fastest in water, somewhat slower
in lettuce leaf decoction, and slowest on a lettuce leaf (62). Conidial
germination usually took place from the end opposite the point of
attachment to the sterigma. In appearance the germ tube was smooth,
hyaline and occasionally single-branched. Tubes often were swollen
where they contacted one another or where they touched conidia,
and at these swellings tubes often branched (85).
The diameter of germ tubes on agar averaged 5 ,; on lettuce
leaves they varied from 2 to 7 i, and their lengths averaged 23 1 before
infection took place. On agar their greatest lengths measured 220 s,
but the longest tubes grew in distilled water, 638 after 24 hours at
160 C (85). Germ tubes were seen to grow for 11 to 24 hours on
any medium, dying after they reached their maximum length (62).
The optimum temperature for germ tube growth was about 150 C,
and this was not influenced by the temperature at which the conidium
was produced (55,85). Optimum temperature for their growth was
generally higher than the optimum for production of their conidia.
No anastomoses have been reported, despite frequent contact
of germ tubes followed by swelling at points of contact. The fact that
germ tubes become swollen when they touch one another or touch
conidia may help to account for some references to appressoria
(85). Publication of photomicrographs and drawings of appressoria
has led the author to believe that those organs were sometimes mis-
named (85). What was referred to as an appressorium, at times
closely resembled a top view of the first spherical vesicle that forms
after the infection hypha enters the epidermal cell lumen (39).
More penetration of stomata by infection hyphae was found
on Romaine than on Bibb lettuce (13). In most cases infection
hyphae in lettuce appeared to penetrate the center of an anticlinal
epidermal cell wall (2). When penetration was sufficiently deep, the
hypha entered the cell lumen. Rarely a hypha grew through the cell
and into an intercellular space without forming a vesicle (39). No
references to the dimensions of these minute infection hyphae were
Approximately 11 hours after a conidium fell upon a lettuce leaf,
an infection hypha had entered the epidermal cell and formed a
thin-walled sphere averaging 16 in diameter. Another vesicle budded
from the sphere, as protoplasm streamed from the conidium into the
new structures. This second vesicle was usually much larger than the
sphere. Its width was similar to the sphere's diameter, but the length
averaged 54 1, (39). Occasionally two of these latter vesicles could
be found in one lettuce epidermal cell; they usually were seen 30
hours after inoculation. These vesicles were produced in the epidermal
cell that was first invaded. On rare occasions a haustorium from the
elongate vesicle penetrated the adjoining epidermal cell wall, where
another elongate vesicle was formed during the ensuing 18 hours. Ap-
parently the original elongate vesicle had derived nourishment from
the epidermal cell, because the latter's protoplasm gradually diminish-
ed until the lettuce cell died, about 72 hours after inoculation (39,
Downy mildew resistant lettuce was penetrated by germ tubes,
smaller vesicles formed, then infection stopped (85).
The first haustoria to develop grew from the secondary vesicle.
They penetrated epidermal cell walls adjoining the initially infected
cell. Later, they protruded from the intercellular mycelium into
parenchyma cells (21, 39).
Haustoria entered cells from all directions. They were club or
flask-shaped and soon became enclosed in a sheath of callose,
deposited by the host cell. Eventually, as the haustorium enlarged,
the originally deposited sheath broke and settled around the base
of the haustorium as a collar. Cursory microscopic examination of such
collars could be misconstrued as optical cross-sections of oospores (21,
39). Possibly this accounts for some of the infrequent reports of
Bremia oospores in lettuce.
There was usually one haustorium per host cell but occasionally
as many as five (85). Their dimensions averaged 9.5 by 15.5 IA. Haus-
toria in lettuce hypocotyls and roots were abnormally small and widely
spaced along the hyphae (39). Hyphae in young leaves were found
to have fewer haustoria than hyphae in older leaves. In the stem
pith of Sonchus oleraceus they were larger and more likely to be
club-shaped than those in lettuce.
All species of the genus Bremia had non-septate, coenocytic,
intercellular mycelium (48, 56, 85). The only exception to the coenocy-
tic condition was the conidiophore, which often had septa in the main
stem and branches (56). Hyphae usually assumed the shape of
intercellular spaces and therefore varied greatly in diameter and
branching; however, the general tendency was to branch dichoto-
mously. Diameters of hyphae varied from 2 to 12 A.
In the hypocotyl and root, hyphae of Bremia lactucae were rela-
tively straight along the host's axis. These hyphae rarely branched
and were of a small but uniform diameter. These unusual, systemic
hyphae had diameters as small as 2-3 a (39). In respect to diameter
they resembled reports of the mycelium of B. graminicola.
Rarely, oogonia and antheridia were found inside host tissues and
crowded together (3, 5, 56). Oogonia had a hyaline, thin, short-lived
outer membrane; they varied in shape, even when located within the
same leaf. In one instance, Lactuca sativa and L. scariola were inocu-
lated and kept under bell jars which were lined with moist filter
papers. Within 10 days oogonia and antheridia were reported to
develop, even before conidia were formed (64).
Oospores usually were spherical, rarely long-elliptical. They av-
eraged 27-30 A in diameter, ranging from 21 to 39 A. Their length-
width ratio usually was 1.25 (64). The outer covering, the epispore,
was thin, pale yellow-brown, transparent, slightly wrinkled, and
rather permanent. Germination of oospores has not been observed.
Oospores were found in .blackened lesions of Senecio vulgaris leaves
(50). Other hosts in which they were found included Lactuca sativa,
L. scariola, Lapsana communis, Leontodon autumnalis, Sonchus ar-
vensis, and S. asper (29, 64). Since B. lactucae cannot be identified
by its oospore, reports of its oospores in susceptible hosts should
be made only after they have been directly connected with the im-
In summation, Bremia lactucae Regel is an obligately parasitic
fungus with no septa in its hyphae, which spreads by means of
air-borne conidia formed on drum-shaped vesicles. Its morphology is
sufficiently unique to permit easy identification by microscopic ex-
Host-parasite relations and fungus organs may be studied through
the use of microtechnic. Leaf lesions or entire small plants were fixed
in 1 part glacial acetic acid to 3 or 4 parts 95% ethanol for 3'/
to 4 hours (85, 91). The liquid was decanted and replaced with Lepik's
lactophenol-alcohol. If left too long in the former fixative, tissues
tended to disintegrate.
Another fixative was used in preparing infected lettuce for
sectioning and staining. It consisted of 5 parts formalin, 5 parts pro-
pionic acid, and 90 parts of 50% ethanol (85). The best cross sections
(12 a) were prepared by dehydrating in tertiary butyl alcohol; they
were mounted in paraffin, sectioned, and stained for 15 hours in 1%
safranin in 95% ethanol (40).
Whole mounts of mildewed lettuce have been cleared, preparatory
to staining, by boiling in glacial acetic acid. Another method for
clearing dead lettuce leaves included soaking in 1 part chloral hydrate
and 1 part phenol (warmed crystals), then a brief soak in 10%
potassium hydroxide solution, followed by a rinse (93). Bremia in
whole lettuce seedlings was readily visible after fixed seedlings were
simmered for 4 to 7 minutes in hot Lepik's lactophenol containing
0.01% orseillin BB (33, 40). Stem and root tangential sections were
prepared in a similar manner.
Observing Bremia in brown, necrotic lettuce leaves was easily
accomplished by fixing in 7% sodium hydroxide in vacuum for 30
minutes. Tissues were then bleached for 15 minutes in 5 6% sodium
hypochlorite. Lettuce tissue was cleared but mycelium retained a brown
color (40). Other successful stains included 1% resorcin blue in dis-
tilled water, aniline blue in lactic acid (21), lactophenol-ethanol con-
taining 0.02 0.2% cotton blue or cotton blue plus 0.1% safranin
(85), Stoughton's thionin and orange-G (75, 85) and cotton blue in
boiling acetic acid (2). If lettuce epidermal cells were infected for
30 hours at a temperature over 150 C, they did not stain with cotton
blue. Normal cells were stained lightly (85).
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U iversity ofFlori