contributions of tyloses and terpenoid aldehyde phytoalexins to verticillium wilt resistance in...
TRANSCRIPT
Physiological Plant Pathology (1978) 12, 1-l 1
Contributions of tyloses and terpenoid aldehyde phytoalexins to Verficilhm wilt resistance in cotton
MARSHALL E. MACE
U.S. Defiartment of Agkulture, Agricultural Research Service, National Cotton Pathology Research Laboratory, P.O. Drawer JF, College Station, Texas 77840, U.S.A.
(Accepted for publication 3uly 1977)
Verticillium wilt-resistant cv. Seabrook Sea Island and -susceptible cv. Rowden cottons were inoculated in the hypocotyl at the ten leaf stage with conidia of Verticillium dahliae. Stem xylem was observed from 10 to 96 h after inoculation for Verticillium hyphal growth and sporulation, vascular occlusion and terpenoid aldehyde synthesis. Rapid occlusion of xylem vessels by tyloses followed by synthesis of fungitoxic terpenoid aldehydes is apparently required for the resistance of Seabrook Sea Island to Verticillium wilt. Occlusion of infected vessels throughout the stem of Seabrook Sea Island by 48 h after inoculation appeared to prevent the systemic distribution of most, if not all,, secondary conidia released at the primary infection sites between 24 and 48 h after inoculation. The fungitoxic terpenoid aldehydes, predominantly hemigossypol and methoxyhemigossypol, then accumulated within the localized primary infection sites. Once V. dahlia was effectively localized, form- ation of new, non-infected xylem tissue apparently enabled Seabrook Sea Island to compen- sate for vessels occluded by responses to the primary infections. Occlusions of infected vessels in the stem of Rowden were delayed. This delay allowed secondary conidia to escape containment at primary infection sites and thereby escape exposure to the localized accumu- lation of terpenoid aldehydes. Because of the extensive systemic distribution of secondary conidia in Rowden, infection of newly formed xylem vessels generally kept pace with their formation, and severe wilt developed.
INTRODUCTION
Wilhelm et al. [30, 311 described the localization of Verticillium albo-atrum (sic) (V. dahliae) in the lower four to five internodes of Verticillium wilt-resistant Go&urn barbadense cottons. When the G. barbadense cultivars Seabrook Sea Island and Waukena White were infected early in the growing season, Verticillium was isolated only from the older or inner xylem of lower internodes in mature plants. The pathogen often colonized a few lower leaves and induced localized wilt, but most of the mature plant showed no wilt symptoms and was generally free of Verticillium. Comparable Gosgpium hirsutum plants failed to restrict the pathogen to the lower stem, and systemic spread of the pathogen and severe wilt ensued.
Prevention of systemic spread of fungal wilt pathogens by rapid occlusion of infected xylem vessels with tyloses has been proposed as a wilt-resistance mechanism in banana [3, 281, hop [24], tomato [4, 23, 271 and ehn [II]; however, certain tomato cultivars forming few or no tyloses after inoculation with the tomato strain of V. albo-atrum were nevertheless highly resistant [IO, 201. The most detailed docu- mentations of the vascular occlusion mechanism were presented by Beckman et al. [Z, 31 for Fusarium wilt of banana and by Talboys [24] for Verticillium wilt of hop.
2 Marshall E. Mace
Occlusion of xylem vessels of banana roots and hop stems with tyloses consistently occurred in advance of fungal colonization in the wilt-resistant cultivars. Although temporary occlusion by gels occurred initially in Fusarium-infected banana roots [3], only subsequent vessel occlusion by tyloses provided a permanent barrier to systemic spread of Fusarium conidia. Vessel occlusion by tyloses was also observed in wilt- susceptible cultivars of banana, but it generally occurred too late to block systemic spread of the conidia.
Phytoalexins may contribute to resistance to fungal wilts. Rishitin was detected in higher concentrations in vascular tissues of wilt-resistant than in wilt-susceptible tomato varieties after infection with Fusarium oxys&orum f. sp. lycopenici [18] or V. albo-atrum [26, 271. The Fusarium wilt-resistant variety showed higher concentrations of rishitin than the susceptible variety 2 days after inoculation, but concentrations in the two varieties were equivalent at 3 days [18]. The terpenoid aldehydes (TA) hemigossypol (1,6,7-trihydroxy-5-isopropyl-3-methyl-8-naph~aldehyde) and 6- methoxyhemigossypol were identified as the major TA phytoalexins induced by V. dahliae infection of the stem xylem of G. barbadense L. cv. Seabrook Sea Island, and G. hirsutum L. cv. Rowden [1.5, 171. Infection of the xylem tissue of cotton stems with V. dahliae induced levels of TA that were toxic to the pathogen; however, no TA was detected in the healthy xylem tissue [S, 321. Synthesis of TA occurred in the diseased xylem tissues of both susceptible and resistant cultivars, but TA accumu- lated more rapidly in the resistant cultivars [S, 321. Bell [6] found that the TA con- centrations of resistant plants were significantly greater than those in the susceptible plants only at 2 to 4 days after inoculation with V. dahliae. Mace et al. [17] demon- strated that the phytoalexins hemigossypol and methoxyhemigossypol were first induced in inoculated stems in scattered, usually solitary, parenchyma cells (contact cells) appressed to the vessel walls in both Verticillium wilt-resistant and -susceptible cottons.
The distribution of fungal vascular pathogens generally occurs by transport of successive generations of conidia in the transpiration stream [3, 221. Therefore, the time required for initial release of secondary conidia in relation to the time required to induce host anatomical and phytoalexin responses could be of critical importance in determining resistance or susceptibility. This paper reports the inter- actions of V, dahliae sporulation and hyphal growth and host tylosis and TA bio- synthesis in wilt-susceptible and -resistant cottons during the first 4 days after inocu- lation and assesses their contributions to susceptibility or resistance to Verticillium wilt.
MATERIALS AND METHODS
Conidia were harvested in sterile distilled water from 7-day-old cultures of Verticillium Miae Kleb. (National Cotton Pathology Research Laboratory non-defoliating strain no. 106) grown at 25 “C on potato-dextrose agar. This strain of V. dahlia is similar to the SS-4 strain described by Schnathorst [21]. Conidia were washed once with sterile distilled water in a centrifuge, suspended in distilled water and standardized with a spectrophotometer (O.D. O-5 at 600 nm = 10’ conidia/ml).
The Verticillium wilt-susceptible cotton cultivar Rowden (G. hirsutum L.) and the resistant cultivar Seabrook Sea Island 12B2 (SBSI G. barbadense L.) were grown in
Verticillium wilt resistance in cotton 3
soil in 20 cm diameter pots to the ten leaf stage in a greenhouse at 25 to 30 “C. The lower hypocotyls (c. 3 cm above the soil line) were then inoculated with c. 0.05 ml of a suspension of conidia (c. 5 x lo6 conidia) at a single site by the hypodermic puncture technique [9]. Plants injected with sterile distilled water served as controls. Plants then were transferred to a controlled environment chamber with a 14 h, 25 “C light (21 000 lx) : 10 h, 18 “C, dark cycle.
Fresh, transverse sections (c. 100 v) for microscopic observations were cut free- hand from the middles of the hypocotyls (c. 2 cm above the inoculation site) and the first six internodes above the hypocotyl. Two sections were cut from each of the seven stem locations in each of four plants at 10, 14, 18, 24, 48, 72 and 96 h and 10 days after inoculation. The experiment was repeated 4 times. Stem sections were mounted in distilled water on a glass slide and vessels were observed micro- scopically for the presence of Verticillium hyphae and conidia, formation of tyloses and occlusion of vessels by tyloses. The number of sections showing hyphae and tyloses were counted and, finally, the number of vessels per section that were occluded with tyloses was recorded.
After the observations for Verticillium growth and tylose formation were com- pleted, the sections were blotted dry and tested for the presence of TA. A saturated solution of SbCl, in 60% HClO, [16] was used to localize histochemically the TA in the fresh, unfixed stem sections. The sections were covered with a few drops of the SbCl, reagent at 23 to 25 “C, covered with a coverslip, allowed to react for 2 min and observed microscopically to determine the extent of the TA response. The per- centage of the stele area showing the red-orange Sb-TA chelate was estimated and assigned grade values of 0 to 5: 0 = trace, 2 = 25%, 3 = 50%, 4 = 75% and 5 = 100%. The red-orange color of the Sb-TA chelates is specific for the TA induced by Verticillium infection of cotton stem stele tissue [17] and the TA in the epidermis and cortex of healthy cotton roots [16]. Additional observations for Verticillium hyphd growth and sporulation were also made at this stage because the red-orange Sb-TA chelate often coats the mycelium and conidia, especially at 2 to 4 days, and makes detection easier. No detailed observations of tyloses were made after treatment with the SbCl, reagent because tyloses frequently were destroyed by the reagent.
Attempts were made to reisolate V. dahlia from each of the seven stem zones at 18 h, 1 to 4 days and 10 days after inoculation. Sections c. O-5 cm long were aseptic- ally removed near the middle of each of the stem zones of plants previously used for microscopic sections. These sections were placed on water agar containing 40 mg/l each of penicillin-G and streptomycin sulfate. The segments were then incubated at 23 to 25 “C for 2 weeks, and the agar was then examined microscopically for the presence of Verticillium.
RESULTS
Fungal growth, TA and tyloses were absent from control plants at all sampling times. Germination of Verticillium conidia and formation of conidiophores were observed in the hypocotyl and first internode of the fresh stem sections of both SBSI and Rowden cottons 10 h after inoculation of the hypocotyl. Conidia had lodged at the scalariform-reticulate perforation plates or end-walls of vessels and grown through the spaces between the secondary wall thickenings to form conidiophores. At 14 h
TABL
E 1
Grow
th an
d sp
orula
tion
of V.
da
hliae
, ind
uctio
n o_
f‘ tqpe
noid
aldeh
ydes
an
d oc
clusio
n of
vess
els
by t
ylose
s in
wilt-s
usce
ptible
Ro
wa’m
an
d -re
sistan
t Se
abro
ok
Sea
Islan
d (S
BSI)
cotto
n ste
ms
18 t
o g6
h a
fh ino
culat
ion
Hyph
al gr
owth@
Sp
orula
tionO
Te
rpen
oid
aldeh
yde*
O Ve
ssels
oc
clude
dC
Posit
ion*
Cultiv
ar
18
24
48
72
96
18
24
48
72
96
18
24
48
72
96
18
24
48
72
96
H SB
SI
+ +
+ +
+ +
+ +
+ +
1.0
2.6
3.3
3.5
3.4
0.0
0.0
5.8
7.1
9.4
Rowd
en
+ +
-I- +
+ +
+ f
+ +
0.0
1.0
2.0
3.0
3.6
0.0
1.6
5.0
18.6
22.4
1 SB
SI
+ +
+ -I-
+ +
+ +
+ +
0.0
1.9
3.1
3.6
3.3
0.0
2.6
13.9
17.7
24.6
Rowd
en
+ +
+ +
+ +
+ +
+ +
0.0
0.0
1.0
2.0
3.0
0.0
2.6
9.1
19.8
26.0
2 SB
SI
++++
+ ----
- 0.0
1-O
2.1
3.2
3.1
0.0
4.8
I 1
.8 23
.9 34
.7 Ro
wden
++
+++
--+++
0.0
0.0
0.1
1.4
2.1
0.0
0.0
4.5
14
.5 20
-o
3 SB
SI
- -
- -
- Ro
wden
-
- +-
+ +
- -
- -
- -
- -
- +
0.0
0.0
1-l
2-2
2.2
0.0
3.3
6.7
21.1
24.4
0.0
0.0
0.0
0.4
1.8
0.0
0.0
0.0
9.4
16.8
4 SB
SI
- -
- -
- -
- -
- _
0.0
0.0
0.0
1.0
1.9
0.0
0.0
4.5
8.3
16.2
Row&
n _
- _
_ +
- _
- _
- 0.0
0.0
0.0
0.0
1.0
0.0
0.0
0.0
5.0
9.7
5 SB
SI
- -
- -
- -
- -
- -
0.0
0.0
0.0
0.0
I.2
0.0
0.0
3.1
4.8
5.9
Row&
n -
- _
- +
- -
_ _
- 0.0
0.0
0.0
0.0
1.0
0.0
0.0
0.0
0.0
3.0
6 SB
SI
- -
- -
- -
- -
- -
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.5
2.6
3.1
Row&
n _
- -
_ -
_ _
- _
_ 0.0
0.0
0.0
0.0
0.4
0.0
0.0
0.0
0.0
0.9
a Mi
crosc
opic
obse
rvatio
n. *
Area
of
stele
cross
-secti
on
show
ing
terpe
noid
aldeh
yde
respo
nse
(red
Sb-te
rpen
oid
chela
te)
: 0,
none
; 1,
trace
; 2,
4; 3,
4; 4,
Q,
c Av
erag
e va
lues/c
ross-s
ectio
n fro
m fou
r ex
perim
ents:
tw
o se
ction
s/stem
po
sition
; fou
r pla
nts
of ea
ch
cultiv
ar
per
expe
rimen
t. d
H,
hypo
cotyl
, 1
to 6
= firs
t to
sixth
inter
node
s ab
ove
hypo
cotyl
.
PLATES 1 to 12. Fresh, freehand sections of stem steles of 6-week-old Rowden and Seabrook Sea Island 12B2 (SBSI) cotton 1 to 10 days after inoculation with V. duhliae. The SbCl, reagent reacted with the disease-induced terpenoid aldehydes (TA) to produce a red Sb chelate of TA. Photographs were taken in the central, most intense areas of the TA response to facilitate comparisons.
PLATE 1. V. duhliae sporulation 24 h after inoculation in hypocotyl of Rowden. TA deposits in vessel wall but not on conidiophores or conidia. x 350. -
PLATE 2. First internode of SBSI 48 h after inoculation showina TA denosits in vessel walls and V. dahlia conidiophores and conidia. x 220.
PLATE 3. Hypocotyl of Rowden 72 h after inoculation showing vessel occluded with tyloses. Heavy and light TA deposits are present on vessel walls and tyloses, respectively. x 220.
PLATE 4. Second internode of SBSI 10 days after inoculation showing vessel occlusion with tyloses coated with heavy deposits of TA. x 220.
PLATE 5. Second internode of SBSI 24 h after inoculation showing TA reaction. TA is restricted largely to point of contact between vessels. x 220.
PLATE 6. First internode of SBSI 48 h after inoculation showing TA localization in contact parenchyma cell (arrow). x 220.
PLATE 7. First internode of Rowden 48 h before inoculation showing TA restricted largely to vessel1 wells.
PLATE 8. First internode of SBSI 48 h after inoculation showing TA. TA has begun to diffuse into parenchyma surrounding vessels. x 145.
PLATE 9. Hypocotyl of Rowden 72 h after inoculation showing TA. Ray parenchyma cells remain largely free of TA. x 145.
PLATE 10. Hypocotyl of SBSI 72 h after inoculation showing TA. Heavy concentrations of TA have diffused into parenchyma surrounding vessels. x 145.
PLATE 11. First internode of SBSI 96 h after inoculation showing heavy deposits of TA in vessel walls and surrounding parenchyma. x 70.
PLATE 12. First internode of Rowden 96 h after inoculation showing TA. Intensity of TA response is similar to that of SBSI in Plate 11. x 70.
lb&- page 41
PLATES 1 t0 12
Verticillium wilt resistance in cotton 5
after inoculation, sections from both lines showed immature conidia only. Conidia were judged to be mature when they were c. 1.5 times the maximum diameter of the conidiophore. No tyloser or TA were seen in either line at 10 and 14 h after inoculation.
Detailed data were presented in Table 1 for periods from 18 to 96 h after inocu- lation. At 18 h, only immature conidia were observed in the hypocotyls and first internodes of SBSI and Rowden. Tyloses, usually small initials, were present in sections from the hypocotyl and first internode of Rowden and in internodes one to three of SBSI. A trace of TA was detected in the xylem of the hypocotyl of SBSI but was absent in Rowden.
Immature and mature-sized conidia (Plate l), TA synthesis and occlusion of vessels by tyloses were observed in both SBSI and Rowden at 24 h after inoculation. Sporulation was observed up to the first internode and hyphal growth up to the second internode in both cultivars. Occlusion of vessels by tyloses was seen only in the hypocotyl and first internode of Rowden, but it occurred as high as the third internode, two internodes in advance of sporulation, in SBSI. Small tyloses, but no occlusion, occurred in the hypocotyl of SBSI. A marked increase in TA synthesis, extending into the second internode (Plate 5), was noted in SBSI, but only a trace of TA was seen in the hypocotyl of Rowden.
At 48 h after inoculation of SBSI, sporulation and hyphal growth still were not observed above the first and second internodes, respectively. In Rowden, sporulation was evident in the second internode. In SBSI, abundant occlusion of vessels by tyloses was seen up to the fourth internode, three internodes in advance of sporulation. Occlusion by tyloses in SBSI occurred in only two to four contiguous vessels in the fifth and sixth internodes. Occluded vessels were generally less numerous in Rowden and were not seen in advance of pathogen sporulation. TA synthesis in SBSI had occurred in xylem tissue as high as the third internode, two internodes in advance of sporulation (Plates 6 and 8) ; however, in Rowden, it was not present above the first internode, one internode below observed sporulation (Plate 7). Deposition of TA on conidiophores and conidia (Plate 2) and tyloses (Plate 3) had begun in the hypocotyl and first internode of SBSI and in the hypocotyl of Rowden.
At 72 h after inoculation, host responses in both cultivars had intensified. Dis- tribution of Verticillium was still restricted to the second internode in SBSI ; however, hyphae were seen as high as the third internode of Rowden. Occlusion of vessels by tyloses occurred consistently in advance of any microscopically observed pathogen growth or sporulation in both lines. In the fifth and sixth internodes, occlusion was absent in Rowden and generally confined to two to four contiguous vessels in SBSI. Occluded vessels were more numerous in SBSI than in Rowden, except in the hypo- cotyl of SBSI where tylose development was sparse. TA synthesis in SBSI (Plate 10) had intensified and now extended into the fourth internode. Although TA had increased in Rowden (Plate 9), it was observed only as high as the third internode.
Verticillium distribution in SBSI at 96 h still was observed only as high as the second internode, unchanged from 24 h after inoculation; however, sporulation and hyphal growth were now observed as high as the third and fourth internodes, respectively, of Rowden. Although more vessels generally were occluded by tyloses in SBSI than in Rowden, occlusion in both cultivars extended from the hypocotyl
6 Marshall E. Mace
into the sixth internode. Tyloses still were sparse in the hypocotyl of SBSI. TA synthesis, as determined by the percentage of the stele area showing the red-orange TA chelate, generally was similar in the two cultivars (Plates 11 and 12).
At 10 days after inoculation, abundant Verticillium mycelial growth and sporul- ation were observed readily throughout the Rowden stems. Rowden exhibited severe wilt, but SBSI was symptomless or occasionally showed wilting in a few lower leaves. No Vertkillium was seen above the first internode in SBSI ; however, the heavy deposits of TA on tyloses (Plate 4) and in unidentified gel-like material in vessels precluded detection of low levels of Verticillium that might have been present in higher internodes. Occlusion of vessels by tyloses was abundant throughout the stems of both Rowden and SBSI, but heavy deposits of TA and the infiltration of the tylose occlusions in many vessels by gelatinous material made accurate counts of vessels occluded with tyloses impossible. TA deposits of grades 5, 4, 4, 4, 3, 2 and 1 in the hypocotyl and the first to the sixth internode, respectively, were noted in both SBSI and Rowden. SBSI had begun to form new, uninfected secondary xylem vessels at 10 days, but such recently formed xylem vessels in Rowden were usually infected.
Stem sections were also examined for occlusion of vessels by gel at the same time that data on pathogen growth, tylosis and TA synthesis were collected. No occlusion by gel was seen in either cultivar up to 48 h after inoculation. At 72 and 96 h, one or two vessels per section were occluded by gel in c. 25% of the sections from the hypocotyl and the first three internodes of SBSI and Rowden. The com- position of the gel is unknown. No gel occurred in controls.
The distribution of the primary inoculum at 18 and 24 h determined by growth of the fungus from stem sections (Table 2) was three or four internodes in advance
TABLE 2
.Nwnber of pkzntso of Seabrook SW Island (SBSI) and Row&n cottonfiom which V. dabliae was r&ok&d at variatu stem &cations a@ stem+unctur~ inoctd&n of the hypocofyl with conidiab
Time after inoculation CUltiVar PositionC 18h 24h 48h 72h 96h lOdays
SBSI H 16 16 16 16 12 0 1 16 16 16 16 16 0 2 16 15 16 16 16 0 3 15 13 13 16 16 1 4 10 11 12 8 9 1 5 3 5 6 8 8 1 6 0 1 2 1 3 0
Rowden H 16 16 16 16 16 16 1 16 16 16 16 16 16 2 16 16 16 16 16 16 3 16 16 16 16 16 16 4 10 15 16 16 16 16 5 4 6 16 16 16 16 6 0 5 16 16 16 16
0 Total of 16 plants : four experiments of four plants per cultivar. b C. 5 x 10’ conidia per plant.‘ 0 H, hypocotyl, 1 to 6 = first to sixth internodes above hypocotyl.
Verticillium wilt resistance in cotton 7
of microscopically detectable Verticillium growth (Table 1) in both SBSI and Rowden. There was a sharp change from sparsity or absence of Verticillium in the fifth and sixth internodes of Rowden at 18 and 24 h to routine isolation of the pathogen from these areas at 48 h and thereafter. This sudden change in the isolation pattern, indicative of the release of secondary conidia, was not observed in the fXth and sixth internodes of SBSI although Verticillium was occasionally isolated from the sixth internode at 48 h and later. Although Verticillium was always isolated from all Rowden stem segments 10 days after inoculation, it was rarely recovered from comparable SBSI stem segments. A prominent yellow halo, only weakly developed around Rowden stem segments, was present around the SBSI stem segments on water agar. Segments from water-injected control plants were free of Verticillium at all times.
DISCUSSION Wilhelm et al. [30, 311 showed that V&killium colonized the roots of wilt-resistant G. barbadense cottons and moved into the lower stem before finally being arrested. The movement of V. dahlia in infected cottons occurs by means of conidia trans- ported in the xylem vessels [13,22]. Talboys [251 suggested that Verticillium inoculum must increase to a critical level before the resistance responses of the host are triggered. A gradual increase in inocuhun probably occurs as V. dahliae moves through the root system of G. barbadense cotton and then invades the lower stem. Eventually, a critical level of inoculum may develop and induce effective resistance responses. The technique of injecting V. akhk inoculum directly into the hypocotyl allows one to eliminate the indeterminate time required for root colonization and secondary inoculum build-up in the lower stem. In such a model system, the exact time of challenge of the intact host can be fixed and the speed, sequence and spatial relation- ships of host and pathogen responses can be determined.
V. a5zhliu.e was occasionally cultured from the fifth and sixth internodes 24 h after inoculation of SBSI and Rowden through the hypocotyl. However, the fimgus was not observed microscopically above the second internode, indicating that very few primary infection sites were initiated in the upper stem. Likewise, when V. &hliaG was introduced through severed tap roots of Rowden and SBSI, Beckman et al. [5J found that 97% of the vessels infected after 24 h occurred in the upper tap root and lower stem within 10 cm of the inoculation site. If we assume similar passage dis- tances for conidia in the present study, 97% of the primary infection sites would be in the hypocotyl and first internode at 24 h. My observations and those of Beckman et al. [a demonstrated that much of the primary conidial inoculum was trapped on the side-walls, reticulate-scalariform end-walls and perforation plates of xylem vessels. The similarity of the primary inoculum distribution patterns in both SBSI and Rowden appears to eliminate anatomical features of healthy xylem as resistance factors. Furthermore, comparable times of germination of primary conidial inoculum of V. dahliae and subsequent sporulation in SBSI and Rowden during the first 24 h after inoculation indicated that no effective inhibition of the pathogen had occurred in either cultivar during this period.
My fungal isolation data (Table 2) indicate that the initial release of secondary conidia of V. aMliae at primary infection sites occurred between 24 and 48 h after inoculation. Garber & Houston [12, 131 also found that Vertkillium conidia were
8 Marshall E. Mace
released and moved to the upper stem and leaves of G. hirsutum cottons within 48 h after hyphal penetration of root vessels.
The rapid occlusion of vessels with tyloses above primary infection sites was apparently the first effective resistance response of the xylem tissue in SBSI. To be effective, xylem vessel occlusions must form before initial release of secondary conidia between 24 and 48 h after inoculation (Table 2). In SBSI, but not in Rowden, occlusion of vessels by tyloses occurred one to four internodes above all micro- scopically observed sporulation at 24 to 72 h. The number of vessels occluded was greater than indicated by the counts because the occlusions were discontinuous within a vessel, and many were not detected in any particular cross-section. Occlu- sion of all infected vessels of SBSI as high as the second internode (c. 15 cm above the hypocotyl inoculation site) at 24 h would isolate and contain 97% of the primary infection sites [5]. The percentage of infected vessels that were occluded could not be determined; however, occlusion by tyloses extended into the third internode in SBSI at 24 h after inoculation. Occlusion of vessels by tyloses at 48 h in SBSI still was limited to the quarter section or to a few contiguous vessels of the stele receiving the primary inoculum. However, occlusion occurred at all seven stem locations and appeared to form rapidly enough to prevent the systemic distribution of most, if not all, of the secondary conidia from the restricted primary infection sites. Occlusions were not detected beyond the second internode of Rowden at 48 h. The microscopic (Table 1) and isolation (Table 2) data indicated essentially no movement ofsecondary conidia in SBSI and their distribution throughout the stem of Rowden.
Talboys [24] found that the occlusion of vessels by tyloses in Verticillium-infected hop was least in the presence of large concentrations of mycelium. He postulated that the concentration of an unidentified fungal metabolite responsible for the induction of tyloses in such areas exceeded the optimum level and inhibited tylosis. Similar sparsity of tyloses and occluded vessels occurred in areas of banana and tomato roots heavily colonized by Fusarium [I]. Vascular occlusion in the heavily infected hypocotyl of SBSI was generally less than that in the hypocotyl of Rowden or in the first three to four internodes above the hypocotyl in SBSI and Rowden. This suggests that an inhibitory level of a “tylose inducer” was synthesized in SBSI but not in Rowden.
Pegg & Selman [19] suggested that IAA induced tylosis in Verticillium-infected tomatoes. IAA was detected in Verticillium cultures and shown to increase in con- centration in Verticillium-infected tomato [19] and cotton [29], but IA4 has not been shown to induce tyloses in these plants. In culture, the banana wilt pathogen Fus- arium oxysporum f. sp. cubense [14] produced IAA, and exogenous IAA induced occlusion of banana root vessels by tyloses [15]. However, IAA was not detected in Fusarium-infected banana roots [M. E. Mace, unpublished].
Gelation is not a significant Verticillium wilt-resistance factor in cotton. Gel occlusions were not detected microscopically in either SBSI or Rowden during the critical 48 h period after inoculation, and they occurred only sparsely at 72 to 96 h. This contrasts with Fusarium wilt of banana [3] where occlusion of vessels by gels preceded occlusion by tyloses as a resistance response. Bugbee [8] suggested that rapid vascular occlusion by gels was in part responsible for the resistance of SBSI to Fusarium wilt.
Verticillium wilt resistance in cotton 9
Bell [6] found by extraction methods that more rapid synthesis of TA phyto- alexins, predominantly hemigossypol and methoxyhemigossypol [7, 17J occurred in SBSI than in Rowden at 2 to 4 days after inoculation with V. duhliae. Fungistatic concentrations of TA phytoalexins occurred in xylem of SBSI stems at 48 h after inoculation [6]. My histochemical data also showed more rapid synthesis of TA in SBSI than in Rowden. However, because of the high sensitivity of the histochemical procedure, differences in the concentrations of TA phytoalexins were detected in SBSI and Rowden as early as 18 h after inoculation (Table 1). Histochemical data (Table 1) also established that the TA phytoalexins were produced after vascular occlusion by tyloses in upper internodes of SBSI and Rowden. At 24 to 72 h after inoculation, occlusion in the upper stem occurred one to two internodes above those showing TA.
At 10 days after inoculation, V. dahliae was rarely cultured from sections of the hypocotyl and the first six internodes of SBSI, but it routinely grew from similar sections of susceptible Rowden. A distinct yellow halo appeared in the agar around all the SBSI sections but it was largely absent around Rowden sections. This may indicate the presence of fungitoxic concentrations of naturally yellow-colored TA; however, the TA phytoalexins, known to be fungistatic to V. duhliue [S, 321, are not known to be fungicidal to the fungus. The TA phytoalexin methoxyhemigossypol constitutes c. 40% of the TA induced in the infected stem xylem of SBSI but only 6% of the TA in Rowden [17]. Methoxyhemigossypol might be a more effective phytoalexin against I’. dahliae than hemigossypol. Thus, TA quality as well as rate of synthesis may contribute to the resistance of SBSI. In addition, hyphal lysis, as demonstrated in tomato infected by V. albo-atrum [lo], may have contributed to the general failure to recover V. duhliue from SBSI stems.
The combined effects of rapid tylosis and TA synthesis are apparently needed for the resistance of SBSI to Verticillium wilt. Rapid occlusion of vessels at primary infection sites in SBSI apparently prevents systemic distribution of secondary conidia and allows subsequent accumulation of TA phytoalexins within the localized primary infection sites. Once V. dahliae is effectively localized in wilt-resistant SBSI, formation of new, non-infected xylem tissue apparently enables SBSI to compensate for vessels occluded by responses to the primary infections. Occlusion of vessels in Rowden is delayed and allows many secondary conidia to escape containment at primary infection sites and thereby to escape exposure to the localized accumulation of TA phytoalexins. Because of extensive systemic distribution of secondary conidia in Rowden, infection of newly formed xylem vessels generally kept pace with their formation, and severe wilt disease developed.
REFERENCES
1. BECKMAN, C. H. (1966). Cell irritability and localization of vascular infections in plants. Phyto- patholoa 56,82 1-824.
2. BECKMAN, C. H. & ~LMOS, S. (1962). Relation of vascular occluding reactions in banana roots to pathogenicity of root-invading fungi. Phyt@athologv 52,893-897.
3. BECKMAN, C. H., HALMOS, S. & MACE, M. E. (1962). The interaction of host pathogen, and soil temperature in relation to susceptibility to Furarium wilt of banana. Phytopathology 52, 134140.
10 Marshall E. Mace
5. BECKMAN, C. H., VAN DER MOLEN, G. E., Mua~mm, W. C. & MACE, M. E. (1976). Vascular structure and distribution of vascular pathogens in cotton. Physiological Plant Pathology 9, 87-94.
6. BELL, A. A. (1969). Phytoalexin production and verticillium wilt resistance. Phytopathology 59, 1119-1127.
7. BELL, A. A., STIPANOVIC, R. D., HOWELL, C. R. & FRYXELL, P. A. (1975). Antimicrobial ter- penoids of Gos&~ium: hemigossypol, 6-methoxyhemigossypol and 6-deoxyhemigossypol. Phyto- &mirtry 14,225-231.
8. BUGBEE, W. M. (1970). Vascular response of cotton to infection by Fusarium oxysfiorum f. sp. vasinfectum. PhytaQathology 68, 121-123.
9. BUGBEE, W. M. & ~LEY, J. T. (1967). A rapid inoculation technique to evaluate the resistance of cotton to Vertkillium albo-atrum. Phyta~atholo~ 57, 1264.
10. DIXON, G. R. & PEGG, G. F. (1969). Hyphal lysis and tylose formation in tomato cultivars infected by Verticillium albo-atrum. Tranractions of fi British Mycological Society 53, 109-l 18.
11. ELDERSMA, D. M. (1973). Tylose formation in elms after inoculation with Ckratocystis ulmi: a possible resistance mechanism. JWh&na?s Journal of Plant Pathology 79, 218-220.
12. GARBER, R. H. & HOUSTON, B. R. (1966). Penetration and development of V.ticillium albo-atrum in the cotton plant. Phytopathology 56,993-l 118.
13. GARBER, R. H. & HO-N, B. R. (1967). Nature of V&izillium wilt resistance in cotton. Phyto- pathology 57,885-888.
14. MACE, M. E. (1965). Isolation and identification of 3-indole-acetic acid from Fusarium oxysporum f. sp. cubcnse. Phytopathology 55, 248-241.
15. MACE, M. E. & SOLIT, E. (1966). Interactions of 3-indoleacetic acid and 3-hydroxytyramine in Fusarium wilt of banana. Phytojathologv 56,245-247.
16. MACE, M. E., BELL, A. A. St STIPANOVIC, R. D. (1974). Histochemistry and isolation of gossypol and related terpenoids in roots of cotton seedlings. Phytojratholotp 64, 1297-1302.
17. MACE, M. E., BELL, A. A. & BECKMAN, C. H. (1976). Histochemistry and identification of disease- induced terpenoid aldehydes in Verticillium wilt-resistant and -susceptible cottons. Canadian 3oumal of Botany 54,2095-2099.
18. MCCAN~E, D. J. & DRYSDALE, R. B. (1975). Production of tomatine and rishitin in tomato plants inoculated with Fusarium oqwporum f. sp. [email protected]. Physialogical Plant Pathology 7, 221-230.
19. PEGG, G. F. & SELMAN, T. W. (1959). An analysis of the growth response of young tomato plants to infection by Vcrticillium albo-atrum. II. The production of growth substances. Annals of Applied Biology 47,222-231.
20. Paoo, G. F. & DIXON, G. R. (1969). The reactions of susceptible and resistant tomato cultivars to strains of Vertic%um albo-atrum. Annals of Applied Biology 63,389-480.
21. SCHNATHOR~T, W. C. & MATHRE, D. E. (1966). Host range and differentiation of a severe form of Verticillium albo-atrum in cotton. Phytokathology 36, 1155-1161.
22. SCHNATHOR~T, W. C., PKUSELY, J. T. & CARNES, H. R. (1968). Rate of movement and bipolar development of Verticillium albo-atrum in cotton plants. In Annual Proceedings of the Beltwide Cotton Productian Research Conf#enct, p. 57 (Abstr.). National Cotton Council, Memphis, TN.
23. SINHA, A. K. & WOOD, R. K. S. (1968). Studies on the nature of resistance in tomato plants to Vcrtkillium albo-atrum. Annals of Applied Bialogy 62,319327.
24. TALBOYS, P. W. (1958). Association of tylosis and hyperplasia of the xylem with vascular invasion of the hop by Verticillium albo-atrum. Transactions of tlu British Mycological So&sty 51, 249-260.
25. TALBOYS, P. W. (1964). A concept of the host-parasite relationship in Verticillium wilt diseases. A’ature 282,361~364.
26. TJAMOS, E. C. & SMITH, I. M. (1974). The role of phytoalexins in the resistance of tomato to Vertkillium wilt. Physiological Plant Pathology 4,249-259.
27. TJAMOS, E. C. & Ssrrrr-r, I. M. (1975). The expression of resistance to Verticillium albo-atrum in monogenically resistant tomato varieties. Physiological Plant Pathologv 6, 215-225.
28. WARDLAW, C. W. (1930). The biology of banana wilt (Panama disease). I. Root inoculation experiments. Annals of Botany 44,741-766.
29. WIESE, M. V. & DEVAY, J. E. (1970). Growth regulator changes in cotton associated with defoli- ation caused by Verticillium albo-atrum. Plant Physiology 45304309.
30. WILHELM, S., SAGAN, J. E. & TIETZ, H. (1970). Seabrook (Giwypium barbadmse) x Rex (Gossypium hirsutum) crosses give Vcrticillium wilt resistant, upland-type all fertile o&pring. In Annual Proceedings of the B.&via? Cotton Production Research Confcrcnce, pp. 70-76. National Cotton Council, Memphis, TN.
Verficillium wilt resistance in cotton 11
31. WILHELM, S., SAOAN, J. E. & TIETZ, H. (1974). Resistance to VcrticiUium wilt in cotton: sources, techniques of identification, inheritance trends, and the resistance potential of multiline cultivars. Phytopathdo~ 64,924931.
32. ZAKI, A. I., KEEN, N. T. & ERWIN, D. C. (1972). Implication of vergosin and hemigossypol in the resistance of cotton to Vertkillium albo-atrum. Phytopathology 62, 1402-1406.