Botanical Studies (2007) 48: 365-375.

*

Corresponding author: E-mail: kowh@dragon.nchu.edu.tw;

Tel: 886-4-22840780; Fax: 886-4-22877585.

INTRODUCTION

The genus of Phytophthora contains several devastating

plant pathogens of worldwide importance. Among them

are late blight of potato caused by P. infestans, black pod

of cacao caused by P. plamivora, and blight and root

rot of peppers caused by P. capsici (Erwin and Ribeiro,

1996). Some species of Phytophthora, such as P. sojae, P.

heveae, and P. katsurae, are homothallic and capable of

producing sexual propagules of oospores in single culture.

Other species such as P. infestans, P. parasitica (= P.

nicotianae) and P. capsici are heterothallic and require the

presence of opposite mating types known as A1 and A2 for

the formation of oospores (Gallegly and Galindo, 1958;

Savage et al., 1968). Some species such as P. megasperma

(Ho et al., 1986a) and P. cinnamomi (Ho et al., 1983) even

contain both homothallic and heterothallic isolates. Among

59 species of Phytophthora listed by Erwin and Ribeiro

(1996), 41 were homothallic and 16 were heterothallic.

The other two species did not form sex organs.

Normal oospores are produced readily by heterothallic

Phytophthora even when opposite mating types of

morphologically and physiologically distinct species are

paired, for example, between the primitive soil-borne P.

cinnamomi and the advanced air-borne P. infestans (Ko,

1980b; Savage et al., 1968). Such an unusual phenomenon

shows the possibility that members of Phytophthora

are unique in the biological world in having no genetic

barrier in crosses involving different species. Another

possible explanation is that chemical stimulation may be

involved in oospore formation during mating, a hypothesis

originally proposed by Ashby (1929).

DISCOVERY OF SEXUAL HORMONES IN

PHYTOPHTHORA

Although several lines of indirect evidence suggest

the involvement of substances stimulatory to sexual

reproduction in heterothallic Phytophthora (Ko, 1980b),

early efforts, including my own, to detect the stimulatory

substances were of no avail (Ko, 1988). Eventually,

unequivocal proof of the production of sexual hormones

was achieved by using a polycarbonate membrane that was

impervious to mycelial growth but allowed free passage of

water-soluble substances, to separate A1 and A2 cultures

and to induce formation of oospores by both cultures (Ko,

1978).

Both A1 and A2 isolates of P. parasitica, P. palmivora,

and P. cinnamomi formed selfed oospores when they were

Hormonal regulation of sexual reproduction in Phytophthora

Wen-Hsiung KO*

Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan

(Received January 15, 2007; Accepted June 23, 2007)

Keywords: £\ hormones; Induction type change; Mitochondrial gene control; Oospore formation; Organelle

transplantation; Phytophthora; Sexual reproduction.

CONTENTS

INTRODUCTION .......................................................................................................................................................... 365

DISCOVERY OF SEXUAL HORMONES IN PHYTOPHTHORA

.............................................................................. 365

GROUPING BASED ON HORMONE PRODUCTION AND RECEPTION

.............................................................. 366

HORMONAL HETEROTHALLISM AND HOMOTHALLISM

................................................................................. 369

FACTORS AFFECTING HORMONAL REGULATION OF SEXUAL REPRODUCTION

....................................... 369

CONVERSION OF INDUCTION TYPE

...................................................................................................................... 369

MITOCHONDRIAL GENE CONTROL OF INDUCTION ("MATING") TYPES

...................................................... 370

NUTRITIONAL SUBSTANCES NEEDED FOR SEXUAL REPRODUCTION ........................................................ 372

FUTURE OUTLOOK .................................................................................................................................................... 372

LITERATURE CITED ................................................................................................................................................... 373

REVIEW PAPER

366

Botanical Studies, Vol. 48, 2007

paired with the other mating type of the same or different

species on the opposite sides of polycarbonate membranes

(Ko, 1978). The successful formation of oospores with the

membrane method demonstrates the stimulation of sexual

reproduction by substances produced by the other mating

type and diffused through the membrane. The sex hormone

produced by A1 isolates of Phytophthora, designatd

hormone £\1, can induce sexual reproduction of A2 but not

A1 isolates. Similarly, sexual reproduction of A1 but not of

A2 isolates can also be induced by hormone £\2 produce by

A2 isolates. Hormonal regulation of sexual reproduction

subsequently has been confirmed with the polycarbonate

membrane method in a number of Phytophthora species

(Table 1), including P. infestans (Shaw et al., 1985;

Shattock et al., 1986; Chang and Ko, 1990a, b; Shaw et al.,

1985), P. capsici (Uchida and Aragaki, 1980), P. cryptogea

(Ho and Jong, 1986), P. drechsleri (Ho, 1986a, b; Shattock

et al., 1986), P. megasperma (Ho, 1986a), P. citrophthora

(Ann, 1984), and P. melonis (Chang et al., 1984).

Homothallic species of Phytophthora also were able

to induce oospore formation of A1 and /or A2 isolates,

when pairings were carried out on the opposite sides

of polycarbonate membranes, indicating their ability

to produce £\ hormones (Table 2). This suggests that,

like heterothallic Phytophthora, sexual reproduction

in homothallic Phytophthora is also controlled by £\

hormones.

Attempts to detect £\ hormones in liquid or solid

cultures of A1 and A2 isolates of P. parasitica were not

successful during the early phase of our study on this

subject. However, when a culture block of P. parasitica

was incubated with a piece of Millipore filter (cellulose

nitrate and cellulose acetate) separated by a polycarbonate

membrane, the filter was able to absorb hormone

produced by the culture and stimulate oospore formation

of the opposite type (Ko, 1983). It was also found that £\

hormones could be extracted from the hormone-loaded

Millipore filters with ethyl ether, and that the isolated

hormones were able to stimulate oospore formation when

they were re-adsorbed on a piece of Millipore filter but not

when they were added directly to agar cultures. Millipore

filter probably can prevent inactivation of the hormones

by agar media. This would explain why oospores were

produced only on the agar medium surface in direct

contact with the membrane when opposite mating types

were paired on the polycarbonate membrane (Ko, 1978).

Molecular weight estimation with the ultrafiltration and

reverse osmosis membranes suggests that both hormones

£\1 and £\2 have molecular weights between 100 and 500

(Ko, 1983).

The best solvents for extracting hormone £\1 adsorbed

on Millipore filter were methylene chloride, hexane, and

95% ethanol, followed by petroleum ether and chloroform

(Chern et al., 1996). For extraction of hormone £\2, 95%

ethanol was the best, followed by methylene chloride,

chloroform, and petroleum ether. Hexane was least

effective in extracting hormone £\2 from the filter. The

observation that hormones £\1 and £\2 were soluble in

organic solvents commonly used for lipid extraction (e.g.

petroleum ether and chloroform), and the stability of both

hormones in acid and base (pH 4-10), suggest that both

hormones £\1 and £\2 are relatively stable, lipid-like natural

products. Hormone £\2 is more polar than hormone £\1

because the former was less soluble in hexane than the

latter, and after partition with hexane or petroleum ether,

hormone £\1 was present only in organic phase while

hormone £\2 was present in both organic and aqueous

phases (Chern et al., 1996).

A method for the large scale extraction of hormones

£\1 and £\2 from agar cultures of A1 and A2 isolates of

P. parasitica, respectively, was subsequently developed

(Chern et al., 1999). For hormone £\1, approximately 120

L of the mixture of chloroform-methanol was used to

extract 1,354 plates (20 L agar medium) of A1 culture, and

for hormone £\2, about 120 L of 95% ethanol was used to

extract 1,296 plates (20 L agar medium) of A2 culture. The

amounts of £\ hormones obtained were sufficient to permit

characterization of their physical and chemical properties,

but inadequate for structural elucidation. Results from this

study suggest that both hormones £\1 and £\2 are neutral

lipids with hydroxyl functional group(s) (Chern et al.,

1999).

All the revealed properties of hormones £\1 mentioned

above were confirmed by the recent report of Qi et al.

(2005). The authors used 1830 L of ethyl acetate to extract

the same volume of liquid culture of A1 isolate of P.

parasitica, and obtained 1.2 mg of pure hormone £\1. Their

analysis showed a diterpene structure with three hydroxyl

functional groups for hormone £\1, which supports the

revelation of £\ hormones as neutral lipids with hydroxyl

functional groups by Chern et al. (1999). According to

the chemical structure, the molecular weight for hormone

£\1 is 344.3, which also confirms the estimated molecular

weights for £\ hormones as between 100 and 500 by Ko

(1983). The chemical structure of hormone £\2 remains to

be elucidated.

GROUPING BASED ON HORMONE

PRODUCTION AND RECEPTION

Based on hormone production and reception, 16

sexuality types, divided into three groups, were proposed

for members of the genus Phytophthora (Ko, 1980a).

Members in Group I do not produce oospores in single

culture. They can release hormones that stimulate others

to produce oospores and /or produce oospores themselves

when stimulated by hormones released by others. Their

sexual reproduction requires cross induction by hormones

and is therefore comparable to those commonly called

"heterothallic." The A1 induction type may belong to

sexuality type S1, S2 or S3, while A2 may belong to S4,

S5 or S6. S7 is not responsive to either hormone £\1 or

£\2 but can stimulate both A1 and A2 to from oospores

while S8 can produce neither hormone £\1 nor £\2 but can

KO ¡X Hormonal regulation of sexual reproduction in

Phytophthora

367

Table 1. Species of heterothallic Phytophthora with hormone-regulated sexual reproduction demonstrated with the polycarbonate

membrane method.

Hormone producer

Oospore producer

Species

Induction Type Species

Induction Type Reference

P. parasitica

A1

P. parasitica

A2

Ko, 1978

2

SA

1

A1

P. palmivora

A2

SA

SA

A1

P. cinnamomi

A2

SA

SA

A1

P. colocasiae

A2

Yu and Chang, 1980

SA

A2

P. parasitica

A1

Ko, 1978

2

SA

A2

P. palmivora

A1

SA

SA

A2

P. cinnamomi

A1

SA

SA

A2

P. melonis

A1

Chang et al., 1984

P. palmivora

A1

P. palmivora

A2

Ko, 1978

SA

A1

P. parasitica

A2

SA

3

SA

A1

P. colocasiae

A2

Yu and Chang, 1980

SA

A2

P. cryptogea

A1

Ho and Jong, 1986

SA

A2

P. drechsleri

A1

SA

SA

A2

P. palmivora

A1

Ko, 1978

SA

A2

P. parasitica

A1

SA

3

P. cinnamomi

A1

P. cinnamomi

A2

Ko, 1978

4

SA

A1

P. parasitica

A2

SA

SA

A1

P. palmivora

A2

SA

SA

A1

P. colocasiae

A2

Yu and Chang, 1980

SA

A1

P. cryptogea

A2

Ho and Jong, 1986

SA

A1

P. drechsleri

A2

SA

SA

A2

P. cinnamomi

A1

Ko, 1978

4

SA

A2

P. parasitica

A1

SA

SA

A2

P. palmivora

A1

SA

SA

A2

P. drechsleri

A1

Ho and Jong, 1986

P. colocasiae

A1

P. colocasiae

A2

Yu and Chang, 1980

SA

A1

P. parasitica

A2

SA

SA

A1

P. palmivora

A2

SA

SA

A1

P. cinnamomi

A2

SA

SA

A2

P. parasitica

A1

SA

SA

A2

P. palmivora

A1

SA

SA

A2

P. cinnamomi

A1

SA

P. infestans

A1

P. infestans

A2

Shen et al., 1983

6

SA

A1

P. capsici

A2

SA

SA

A1

P. palmivora

A2

SA

SA

A1

P. parasitica

A2

SA

SA

A1

P. drechsleri

A2

Shattock et al., 1986

8

SA

A2

P. infestans

A1

Chang and Ko, 1990b

7

P. capsici

A1

P. capsici

A2

Uchida and Aragaki, 1980

SA

A2

SA

A1

SA

P. citrophthora

A1

P. parasitica

A2

Ann, 1984

P. megasperma

A1

P. megasperma

A2

Ho, 1986a

368

Botanical Studies, Vol. 48, 2007

be stimulated by both A1 and A2 to form oospores. S7

and S8 are A1, A2 induction type. Members in Group II

produce oospores in single culture and are comparable

to those commonly called "homothallic" or A1A2 type.

They form oospores by self-induction induced by self-

produced hormones and are designated A1A2 induction

type. This group includes S9 to S15. Those isolates

belonging to Group III are neuter and are designated AO.

They cannot stimulate others nor be stimulated to produce

oospores. Only S16 belongs to this group. Prior to 1988,

only S1, S2, S4, S5 and S16 have been found in species

of Phytophthora (Ko, 1988). An unidentified species of

Phytophthora belonging to S16 also was isolated from a

diseased strawberry stem tissue (Chang, 1988).

Rubin and Cohen (2006) recently reported the isolation

of "unusual mating type" of P. infestans. These isolates

were sterile when inoculated alone but produced oospores

in tomato tissue when co-inoculated with and A1 or A2

isolate. According to the grouping based on hormonal

regulation mentioned above, the sexual behavior of

these isolates are not unusual. They fit the description of

sexuality type S8 of the A1, A2 induction type.

Table 2. Species of homothallic Phytophthora with hormone-regulated sexual reproduction demonstrated with the polycarbonate

membrane method.

Hormone producer

Oospore producer

Species

Induction Type

Species

Induction Type

Reference

P. heveae

A1A2

P. parasitica

A1

Ko, 1980a

P. cactorum

A1A2

SA

1

A1

SA

P. katsurae

A1A2

SA

A1

SA

P. humicola

A1A2

SA

A1

Ko and Ann, 1985

P. citricola

A1A2

SA

A1

Ko et al., 1982

P. melonis

A1A2

SA

A1

Chang et al., 1984

P. heveae

A1A2

SA

A2

Ko, 1980a

P. sojae

A1A2

SA

A2

SA

P. insolita

A1A2

SA

A2

Ann and Ko, 1980

P. cinnamomi

A1A2

P. cinnamomi

A2

Ho et al., 1983

1

SA= the same as above.

Hormone producer

Oospore producer

Species

Induction Type Species

Induction Type Reference

SA

A2

SA

A1

SA

P. melonis

A1

P. parasitica

A2

Chang et al., 1984

P. drechsleri

A1

P. drechsleri

A2

Ho, 1986b

SA

A2

SA

A1

SA

SA

A2

P. infestans

A1

Shattock et al., 1986

8

P. cryptogea

A1

P. cryptogea

A2

Ho and Jong, 1986

SA

A2

SA

A1

SA

P. drechsleri

A1

P. drechsleri

A2

SA

SA

A2

SA

A1

SA

1

SA= the same as above.

2

Also reported by Ko et al., 1986; Chang and Ko, 1990a; Ko and Kunimoto, 1981; Ann, 1984.

3

Also reported by Ko and Kunimoto, 1981; Ann, 1984.

4

Also reported by Ann and Ko, 1989; Ho et al., 1983.

5

Also reported by Yu et al., 1981.

6

Also reported by Chang and Ko, 1990b; Shaw et al., 1985.

7

Also reported by Shattock et al., 1986; Shaw et al., 1985.

8

Also reported by Skidmore et al., 1984.

Table 1. (Continued.)

KO ¡X Hormonal regulation of sexual reproduction in

Phytophthora

369

HORMONAL HETEROTHALLISM AND

HOMOTHALLISM

Genetic exchange in the pairings between A1 and A2

types of different species of cross-inducing Phytophthora

has been shown to be essentially nonexistent (Boccas and

Zentmyer, 1976; Boccas, 1981; Erselius and Shaw, 1982;

Chang and Ko, 1993). Consequently, all sexual progenies

are produced by either A1 or A2 type by selfing induced

by £\ hormones (Figure 1A). However, both hybrid and

self progenies of A1 and A2 types are produced following

induction by £\ hormones when isolates of the same

species are paired (Chang and Ko, 1990a; 1992; Judelson,

1997) (Figure 1B). During the sexual reproduction of self-

inducing Phytophthora, self progeny of A1A2 type is

produced following induction by £\ hormones (Figure 1C).

Requirement of the presence of an opposite mating type

in sexual reproduction of "heterothallic" Phytophthora

is for production and reception of mating-type-specific

hormones to initiate the process of selfing or hybridization.

"Homothallic" species of Phytophthora do not require the

presence of another mating type in sexual reproduction

because they possess receptor(s) for self-produced £\

hormone(s) to initiate selfing. This is a novel mode

of sexual reproduction in the biological world. It is a

chemical heterothallism and homothallism, rather than the

conventional biological heterothallism and homothallism.

FACTORS AFFECTING HORMONAL REGULATION

OF SEXUAL REPRODUCTION

The influence of light on the sexual reproduction

o f Phytophthora is well documented (Harnish, 1965;

Klisiewicz, 1970). Few or no oospores were found under

continuous light while many were produced in darkness

(Chang and Shu, 1988; Wang et al., 2005; Ko et al., 2006).

A1 and A2 isolates of P. parasitica were exposed to light

at different stages of sexual development to study the

mode of action of light on hormonal regulation of sexual

reproduction (Chern and Ko, 1993). Light was found to

be inhibitory to the production of £\ hormones but not to

the formation of the receptors of these hormones. After

being produced, £\ hormones were stable under light.

Light was also inhibitory to hormone induction of sexual

reproduction but not oospore formation after hormone

induction. Using A2 isolate of P. colocasiae as a hormone

producer and A1 isolate of P. parasitica as a hormone

receptor, Yu et al. (1981) also showed that hormone

production, but not oospore formation, after hormone

induction was greatly inhibited by light.

For most species of Phytophthora tested, the maximum

temperature permiting vegetative growth is always

inhibitory to sexual reproduction (Chern and Ko, 1994).

Although both A1 and A2 isolates of P. parasitica were

capable of growth at 34¢XC, no oospores were produced

when they were paired at this temperature. These two

isolates were, therefore, exposed to 34¢XC at different

stages of sexual development to study the mode of action

of high temperature on hormonal regulation of sexual

reproduction (Chern and Ko, 1994). The production of

£\ hormones and their receptors was inhibited at high

temperature. However, after being produced, £\ hormones

and their receptors were stable at high temperature. No

oospores were produced when cultures were exposed to

high temperature during hormone reception, or oospore

formation after hormone induction.

CONVERSION OF INDUCTION TYPE

In 1981, the frequently observed phenomenon of

oospore sectors in colonies originating from aged cultures

of A1 or A2 isolates of Phytophthora was found to be due

to the appearance of the opposite induction type during

long term storage (Ko, 1981). During the same period, it

was found that the fungicide chloroneb also could cause

A2 isolate of P. capsici to change induction type and form

oospore sectors on agar medium (Ko, 1981). Subsequently,

other fungicides and the antibiotic streptomycin also

were reported to have the ability to cause conversion of

induction types (Table 3). Progeny derived from selfed

oospores of the A1 or A2 induction type of P. parasitica

(Ann and Ko, 1988) and P. infestans (Ko, 1994) induced

by £\ hormones consisted of the parental induction type

and the new opposite type, indicating the ability of selfing

to cause conversion of induction type.

Some propagules of the A1 and A2 isolates of P.

parasitica were converted to A1A2 type after long-term

Figure 1. Schematic illustration of hormonal regulation of sexual reproduction in Phytophthora. (A), cross induction of different

species; (B), cross induction of the same species; and (C), self induction.

370

Botanical Studies, Vol. 48, 2007

storage and exposure to chlorneb, respectively (Ko, 1981).

Selfing, segregation and certain fungicides also can cause

conversion of A1 and A2 isolates of Phytophthora t o

A1A2 type (Table 4). This kind of A1A2 type is usually

unstable, and some of its zoospore propagules may be

converted to A1 or A2 again (Mortimer et al., 1977; Ko,

1981; Chang and Ko, 1990b).

When self-inducing (A1A2) P. boehmeriae was grown

on medium containing the fungicide ethazol, the organism

produced oospore-free sectors from which self-sterile

cultures were obtained (Zhou et al., 1997). Single zoospore

cultures derived from these self-sterile cultures were of

the A1A2, A1, A2 and A0 types. So far this is the only

example of a self-inducing species of Phytophthora being

converted to cross-induction.

Conversion of induction type in cross-induing

Phytophthora is a reversible process (Ko, 1981; Ko et al.,

1986). When an induction type is converted to the opposite

type, both hormone production and reception are changed

(Ko, 1981; Ko et al., 1986). This indicates that genes for

production of hormone £\1 (P1) and reception of hormone

£\2 ( R2) in A1 are linked and genes for production of

hormone £\2 ( P2) and reception of hormone £\1 (R1) in A2

also are linked, and that A1 (P1R2) and A2 (P2R1) genes

are on the same chromosome. To account for the reversible

nature of induction type conversion, it is further postulated

that the transcription of such linked genes is regulated by a

repressor that represses the expression of A2 (P2R1) gene

in A1 induction type with one molecular configuration

and A1 (P1R2) genes in A2 induction type with another

configuration. Based on this hypothesis, the A1A2

induction type has either an inactive repressor or two

chromosomes, one with A1 (P1R2) genes repressed and

another with A2 (P2R1) genes repressed. The A0 induction

type contains either a super repressor capable of repressing

both A1 (P1R2) and A2 (P2R1) genes concomitantly, or a

chromosome lacking the A1 (P1R2) and A2 (P2R1) genes,

according to the same hypothesis.

MITOCHONDRIAL GENE CONTROL OF

INDUCTION ("MATING") TYPES

Induction types in Phytophthora have been considered

to be determined by either nuclear genes or cytoplasmic

factors (Timmer et al., 1970). Based on cytological

observations and segregation pattern, it was proposed that

the A1 induction type is homozygous recessive aa and A2

is heterozygous Aa, while the self inducing A1A2 type is a

trisomic Aaa (Mortimer et al., 1977; Sansome, 1980). This

hypothesis was refuted by the observation of the induction

of segregation of A2 and A1A2 from A1 by aging and

chemical treatments (Ko, 1981; Ann and Ko, 1989;

Chang and Ko, 1990b) because the homozygous recessive

character should not segregate. Most of the data indicating

nuclear inheritance of mating type were based on genetic

mapping studies involving either the use of nuclear DNA

Table 3. Factors causing conversion of induction type A1 to A2 and vice versa in Phytophthora.

Type of conversion

Species

Causal factor

Reference

A1 ¡÷ A2

P. parasitica

Aging

Ko, 1981

Selfing

Ann and Ko, 1988

Ethazol

Ko et al., 1986

Streptomycin

Ann and Ko, 1992

Metalaxyl

Chang and Ko, 1990

Chloroneb

Ann and Ko, 1989

P. cinnamomi

Ethazol, chloroneb

Ann and Ko, 1989

P. infestans

Selfing

Ko, 1994

Metalaxyl

Chang and Ko, 1990

Mefenoxam

Groves and Ristaino, 2000

A2 ¡÷ A1

P. parasitica

Aging

Ko, 1981

Selfing

Ann and Ko, 1988

Chloroneb

Ko et al., 1986

Streptomycin

Ann and Ko, 1992

Ethazol

Ann and Ko, 1989

P. cinnamomi

Ethazol

Ann and Ko, 1989

P. infestans

Selfing

Ko, 1994

Metalaxyl

Chang and Ko, 1990

Mefenoxam, benomyl

Groves and Ristaino, 2000

KO ¡X Hormonal regulation of sexual reproduction in

Phytophthora

371

markers for the construction of genetic linkage maps

(Van der Lee et al., 2004) or the search for induction type

linked nuclear DNA markers (Judelson, 1996). Their data,

which were based only on association, do not prove that

induction type is a nuclear trait. The putative induction

type locus still has not been cloned.

Segregation in colony morphology during conidial

propagation of a culture originating from a single

conidium, referred to as a "dual phenomenon" in

imperfect fungi, was found to be due to the dissociation

of multinucleate organisms in a heterokaryotic condition

(Hansen, 1938). Zoospores produced by an A2 culture of

P. drechsleri, which originated from a zoospore, gave rise

to A1, A2 and A1A2 cultures (Mortimer et al., 1977). The

single-zoospore A1A2 cultures of P. parasitica (Ko, 1981)

and P. infestans (Chang and Ko, 1990b) also produced A1

and A1A2 zoospore cultures. Such phenomena cannot be

explained as the result of dissociation of heterokaryons

because most zoospores are uninucleate (Mortimer et

al., 1977; Zheng and Ko, 1997). It is conceivable that

induction types are controlled by different mitochondrial

DNA. Both A1 and A2 mitochondria probably exist

in each isolate of Phytophthora, and the expression of

induction type is the end result of the interaction between

these two types of mitochondria, similar to the behavior of

antibiotic resistance in Phytophthora (Gu and Ko, 2000c).

Recently, we developed methods for protoplast fusion (Gu

and Ko, 1998; 2000a), nuclear transplantation (Gu and Ko,

1998; 2000b), mitochondrial transplantation (Gu and Ko,

2000c), and isolation of single transplantation products

(Ho and Ko, 1997). These techniques were used to test if

induction type genes are located in nuclei or mitochondria.

Chang and Ko (1990b) showed that metalaxyl resistance

(M

r

) and chloroneb resistance (Cn

r

) in P. parasitica were

each conferred by a nuclear gene while streptomycin

resistance (S

r

) and chloramphenicol resistance (Cp

r

) were

each conferred by a cytoplasmic gene. When protoplasts

carrying M

r

nuclei from A1 isolate of P. parasitica were

fused with protoplasts carrying Cn

r

nuclei from A2 isolate

of the same species, fusion products carrying M

r

nuclei

were either the A2 or A1A2 type while those carrying Cn

r

nuclei were either the A1, A2, or A1A2 type (Gu and Ko,

2005). Fusion products carrying M

r

and Cn

r

nuclei also

behaved as either an A1, A2 or A1A2 type. The result

refutes the hypothesis that induction types in Phytophthora

are controlled by nuclear genes. When nuclei from A1

isolate of P. parasitica were fused with protoplasts from

the A2 isolate of the same species and vice versa, all the

Table 4. Factors causing conversion from cross induction to self induction and vice versa in Phytophthora.

Type of conversion

Species

Causal factor

Reference

A1 ¡÷ A1A2

P. parasitica

Aging

Ko, 1981

Selfing

Ann and Ko, 1988

P. infestans

Selfing

Ko, 1994

Metalaxyl

Chang and Ko, 1996b

P. cinnamomi

Ethazol, chloroneb

Ann and Ko, 1989

A2 ¡÷ A1A2

P. parasitica

Chloroneb

Ko, 1981

Selfing

Ann and Ko, 1988

P. cinnamomi

Ethazol

Ann and Ko, 1989

P. drechsleri

Segregation

Mortimer et al., 1977

A1A2 ¡÷ A1

P. parasitica

Segregation

Ko, 1981

P. infestans

Segregation

Chang and Ko, 1990b

1

P. drechsleri

Segregation

Mortimer et al., 1977

P. boehmeriae

Ethazol

Zhou et al., 1997

A1A2 ¡÷ A2

P. parasitica

Segregation

Ko, 1981

P. infestans

Segregation

Fyfe and Shaw, 1992

P. drechsleri

Segregation

Mortimer et al., 1977

P. boehmeriae

Ethazol

Zhou et al., 1997

A1A2 ¡÷ A0

P. boehmeriae

Ethazol

Zhou et al., 1997

1

Also reported by Fyfe and Shaw, 1992.

372

Botanical Studies, Vol. 48, 2007

nuclear hybrids expressed the induction type characterstics

of protoplast parent. The same was true when the nuclei

from the A1 isolate of P. parasitica were fused with

protoplasts from the A0 isolate of P. capsici and vice

versa. These results confirm the observation that induction

type genes are not located in the nuclei and suggest the

presence of induction type genes in the cytoplasms of the

recipient protoplasts. When mitochondria from the A1

isolate of P. parasitica were fused with protoplasts from

the A2 isolate of the same species, the induction type of

three out of five regenerated protoplasts was changed from

A2 to A1 type. The result demonstrated the decisive effect

of the sexuality of mitochondrial donor on induction type

characterstics of mitochondrial hybrids and suggested

the presence of induction type genes in mitochondria. All

of the mitochondrial hybrids resulting from the transfer

of mitochondria from the A0 isolate of P. capsici into

protoplasts from A1 isolate of P. parasitica were all of

the A0 type. The result further supports the hypothesis of

the presence of induction type genes in mitochondria in

Phytophthora.

NUTRITIONAL SUBSTANCES NEEDED FOR

SEXUAL REPRODUCTION

Prior to 1983 when Ko and Ho (1983) reported

the discovery that lecithin was stimulatory to oospore

formation of P. parasitica and P. cactorum, sterols were

the only nutritional substances known to be beneficial

to sexual reproduction in Phytophthora (Ko, 1998).

Subsequently, cephalin was reported stimulatory to

oospore formation of P. cactorum (Ko, 1985). In 1997,

Jee et al. (1997) found that the 99% pure commercial

lecithin from one of the shipments was partially inhibitory

to the growth of P. cactorum on basal medium and was

not stimulatory to oospore formation of the fungus. When

it was dissolved in ether and then washed with deionized

water or NaCl solution, the inhibitory effect disappeared,

and the compound became strongly stimulatory to oospore

formation. After removal of similar inhibitory substance,

a number of fatty acids and related compounds became

stimulatory to oospore formation of Phytophthora (Jee and

Ko, 1997; Jee et al., 2002; Wu et al., 2003).

Previously, failure of Phytophthora to produce oospores

in basal medium was believed to be due to their inability to

synthesize the substances required for sexual reproduction

(Hendrix, 1970; Elliott, 1983). However, when mycelium

of P. cactorum produced in liquid basal medium was

transferred to nutrient-free water agarose, oospores were

produced (Jee and Ko, 1998). The organism also produced

oospores on solid basal medium near the edge of the plates

after a prolonged incubation period. These results showed

that P. cactorum does not need special exogenous nutrients

for sexual reproduction but does require a stress factor like

nutrient deprivation or aging to trigger the sexual process.

Furthermore, extracts from the mycelia of a Phytophthora

species grown in liquid basal medium were found to be

stimulatory to their own sexual reproduction, indicating

their ability to synthesize substances needed for sexual

reproduction (Jee and Ko, 1998; Wu et al., 2003).

FUTURE OUTLOOK

Following the detailed characterization of chemical and

physical properties of £\ hormones (Ko, 1983; Chern et al.,

1996; 1999), the chemical structure of hormone £\1 was

revealed (Qi et al., 2005) although hormone £\2 remains to

be determined. Hormone £\2 appears to be more difficult

to work with (M. Ojika, Nagoya University, Personal

communication). The following information may be useful

for future elucidation of the chemical structure of hormone

£\2. Solvent different from that used for extraction of

hormone £\1 probably should be used. According to tests

with £\ hormones adsorbed on Millipore filter, 95% ethanol

was best for extraction of hormone £\2, followed by

methylene chloride (Chern et al., 1996). For detecting the

activity of hormone £\2 in an extract, the hormone should

be adsorbed on a piece of Millipore filter composed of

cellulose nitrate and cellulose acetate (Ko, 1983; Chern

et al., 1996). Ability of hormone £\2 adsorbed on this type

of Millipore filter to induce oospore formation increased

about 25 fold in comparison with the same amount of

hormone added directly to the receptor culture (Chern

et al., 1996). It is also important to know that although

hormones £\1 and £\2 have several similar characteristics,

hormone £\2 has a polarity greater than that of hormone

£\1, and is too polar to be analyzed by gas chromatography

(Chern et al., 1999).

The discovery of control of induction types by

mitochondrial genes in Phytophthora points to a new

direction for research on genetics of sexual reproduction

in this group of organisms. Reversible conversion of

induction types led to the hypothesis that both A1 and A2

induction types contain A1 and A2 genes, and that the

expression of A1 induction type is the result of repression

of A2 gene by a repressor referred to as A2 repressor and

the expression of A1 gene by another repressor referred

to as A1 repressor (Ko, 1981; 1988). The other possibility

is that both A1 and A2 induction types contain two kinds

of mitochondrial DNA, one with A1 gene (A1 mtDNA)

and another with A2 gene (A2 mtDNA). Based on this

hypothesis, A1 induction type has A1 mtDNA as the

predominant type and A2 mtDNA as the minor type, and

A2 induction type is just opposite, having A2 mtDNA as

the predominant type and A1 mtDNA as the minor type,

a system similar to that proposed for mitochondrial gene

control of antibiotics (Gu and Ko, 2000c). The validity of

each hypothesis remains to be tested.

Relatively little is known about hormonal regulation

of sexual reproduction in the closely related genus of

Pythium. Using the polycarbonate membrane technique,

hormonal regulation of sexual reproduction has been

demonstrated in heterothallic Pythium splendens (Guo

and Ko, 1991). Aging (Guo and Ko, 1991) and selfing

(Guo and Ko, 1996) also caused mating type change in

Py. splendens, and lecithins were found to be stimulatory

KO ¡X Hormonal regulation of sexual reproduction in

Phytophthora

373

to sexual reproduction in Pythium aphanidermatum (Ko,

1986). Whether similar events also occur in other species

of Pythium remains to be investigated.

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