DSpace at VNU: Four new species of Kockovaella isolated from plant leaves collected in Vietnam

DSpace at VNU: Four new species of Kockovaella isolated from plant leaves collected in Vietnam tài liệu, giáo án, bài gi...

Kockovaella, a ballistoconidiogenous anamorphic

yeast genus, was described by Nakase et al (1991)

This genus is characterized by the reproduction of

bal-listoconidia, nonballistosporous stalked conidia and

budding cells, Q-10 as the major ubiquinone, and the

presence of xylose in the cells Six species are

recog-nized in the genus: K imperatae, K thailandica

(Nakase et al., 1991), K sacchari (Takashima and

Nakase, 1998), K machilophila, K phaffii, and K

schi-mae (Cañete-Gibas et al., 1998)

During a survey of ballistoconidiogenous yeasts on plant materials of Asia, five yeasts isolated from

Viet-nam were assigned to the genus Kockovaella Four

new species are proposed for these five strains based

on analyses of 18S rDNA sequence, sequences of in-ternal transcribed spacer (ITS) regions, and DNA-DNA reassociation experiments

Materials and Methods

Yeast strains The yeast strains used in this study

were isolated from plant leaves collected in Cuc Phuong National Forest of Ninh Binh, Vietnam (Table 1), using the ballistoconidium-fall method on YM agar

as reported by Nakase and Takashima (1993)

Morphological, physiological, and biochemical char-acteristics. Most methods used for the examination

of morphological, physiological, and biochemical

char-Four new species of Kockovaella isolated from plant

leaves collected in Vietnam

Dao Thi Luong,1,2,* Masako Takashima,1Pham Van Ty,2 Nguyen Lan Dung,2and Takashi Nakase1,†

Wako, Saitama 351–0198, Japan

Vietnam National University, Hanoi, Vietnam

(Received June 26, 2000; Accepted November 9, 2000)

Five ballistoconidiogenous yeast strains, isolated from plant leaves at Cuc Phuong National

For-est of Ninh Binh, Vietnam, were assigned to the genus Kockovaella based on morphological and

chemotaxonomical characteristics They represent four new species based on analyses of 18S

rDNA sequence, sequences of internal transcribed spacer regions, and DNA-DNA reassociation

experiments Four new species, Kockovaella calophylli (1 strain), Kockovaella cucphuongensis

(2 strains), Kockovaella litseae (1 strain), and Kockovaella vietnamensis (1 strain) are proposed

for these strains.

Key Words——Kockovaella calophylli; Kockovaella cucphuongensis; Kockovaella litseae; Kockovaella

vietnamensis; systematics

* Address reprint requests to: Ms Dao Thi Luong, Vietnam

Type Culture Collection, Centre of Applied Microbiology,

Univer-sity of Science, Vietnam National UniverUniver-sity, Hanoi, Vietnam.

† Present Address: Yothi Research Unit, National Center for

Genetic Engineering and Biotechnology (BIOTEC), National

Science and Technology Development Agency, 73/1 Rama VI

Road, Bangkok 10400, Thailand.

Full Paper

acteristics were described by Yarrow (1998) The

de-termination of maximum growth temperature was

made in YM broth, using metal block baths The

as-similation of nitrogen compounds was determined by

the method of Nakase and Suzuki (1986) The vitamin

requirement followed the method by Komagata and

Nakase (1967)

Chemotaxonomic characteristics. Extraction,

pu-rification, and identification of ubiquinones were

car-ried out according to the method of Nakase and

Suzuki (1986) The presence or absence of xylose in

the cells was analyzed by thin-layer chromatography

(Nakase et al., 1976) after hydrolizing the cells with

tri-fluoroacetic acid (Suzuki and Nakase, 1988)

Sequencing and phylogenetic analysis. The

se-quences of 18S rDNA and ITS regions, including 5.8S

rDNA, was determined after amplifying the DNA by

using PCR Both strands were sequenced directly

(Takashima and Nakase, 1999) Generated sequences

were aligned with related species by using the

CLUSTAL W ver 1.74 computer program (Thompson

et al., 1994) Reference sequences used for the

phylo-genetic study were obtained from the database (Table

2) The phylogenetic tree was constructed from the

evolutionary distance data according to Kimura (1980)

by use of the neighbor-joining method (Saitou and Nei,

1987) Sites where gaps existed in any sequences

were excluded Bootstrap analyses (Felsenstein,

1985) were performed from 1,000 random

resam-plings For a comparison of ITS regions among closely

related species, pairwise sequences were aligned by

sight, and the sequence similarity including gaps was

calculated

DNA-DNA relatedness. Isolation and purification of DNA used the methods of Takashima and Nakase (2000) The DNA base composition was determined by HPLC after the enzymatic digestion of DNA to deoxyri-bonucleosides (Tamaoka and Komagata, 1984) The DNA-GC Kit (Yamasa Shoyu Co Ltd., Chiba, Japan) was used as a quantitative standard DNA-DNA reas-sociation experiments used a membrane-filter method (Hamamoto and Nakase, 1995)

Results and Discussion

Five isolates (VY-61, VY-67, VY-I25, VY-127, and VY-137) were characterized by the presence of xylose

in the cells, Q-10 as a major ubiquinone, the produc-tion of symmetrical ballistoconidia, the proliferaproduc-tion by stalked conidia, and budding cells The isolates were

identified in the genus Kockovaella (Nakase et al.,

1991)

The 18S rDNA sequences of five Kockovaella

iso-lates were determined and a phylogenetic tree for

these isolates and 45 species of the genera Bullera, Cryptococcus, Fellomyces, and Kockovaella was

con-structed by the neighbor-joining method based on 1,674 bases (Fig 1) The result showed that five

iso-lates were in a cluster and closely related with Fel-lomyces chinensis (Prillinger et al., 1997), F distylii (Hamamoto et al., 1998), F lichenicola, F sichuanen-sis (Prillinger et al., 1997), and Kockovaella schimae.

The sequences of ITS regions were determined and the sequence similarity was calculated between five

Table 1 Strains used in this study.

DDBJ accession numbers

18S rDNA ITS & 5.8S

T Type strain.

isolates of the genus Kockovaella and phylogenetically

closely related species (Table 3) The results showed

that those of VY-67 and VY-125 were identical and

showed 97.6–99.1% sequence similarity for ITS1 and

97.1–99.1% for ITS2 between other isolates Those of

VY-61, VY-127, and VY-137 showed 99.1–100%,

97.1–99.1%, and 97.1–100% sequence similarity for

ITS1 and 96.5–98.2%, 97.1–99.2%, and 99.1–99.2%

for ITS2, respectively, between other isolates They

showed less than 90% similarity for ITS1 and 86.8–

96.4% for ITS2 between closely related species

These results indicated that Kockovaella strains

iso-lated from plants of Vietnam were distinct from known

Fellomyces and Kockovaella species, and they were

closely related to each other (Sugita et al., 1999) ITS

sequence data also indicated that F chinensis and F.

lichenicola were phylogenetically closely related For

the two species, we will discuss another paper

DNA-DNA reassociation experiments were carried

out among five isolates and phylogenetically related

species (Table 4) Two strains VY-67 and VY-125

showed a high DNA relatedness with each other

(84–93%) and low reassociation values between other isolates (33–47%) and to reference species (2–17%) Strains VY-61 and VY-127 showed low reassociation values between other isolates (31–35% and 15–28%, respectively) and to reference species (2–16% and 2–10%, respectively) Strain VY-137 showed 52–73% relatedness with other isolates at 67°C, when DNA of VY-137 was employed as a probe, whereas it showed 25–47% relatedness when those of other isolates were employed as probes When the experiments were car-ried out at 69°C, DNA relatedness between VY-137

and other Kockovaella isolates were a little lower than

those at 67°C (31–56%) The experiments performed again at 74°C, using the same filters, showed the low reassociation values between other isolates (33–44%) These results indicated that the loosely bound DNA on the filters at 67°C or 69°C was released at an elevated temperature Based on the facts, two strains, VY-67 and VY-125, were considered to represent a single new species, and strains VY-61, VY-127, and VY-137 represented three different new species

Because these four new species are closely related

Table 2 18S rDNA sequence data obtained from the databank for constructing the phylogenetic tree.

as described above and shown in Table 5, however,

they can distinguish from each other by the

combina-tion of assimilacombina-tion ability of glycerol, erythritol, ribitol,

2-ketogluconic acid, DL-lactic acid, and inositol They are also discriminated from each other by ITS se-quences (Fig 2, A and B)

Fig 1. Phylogenetic tree of five isolates of the genus Kockovaella and related species based on the 18S rDNA sequences

The tree was constructed from the evolutionary distance data according to Kimura (1980), using the neighbor-joining method (Saitou and Nei, 1987) with bootstrapping (Felsenstein, 1985) The numerals represent results from 1,000 replicate bootstrap sam-plings (a frequency of less than 50% is not shown)

Description of New Taxa

Kockovaella calophylli Luong, Takashima, Ty, Dung

& Nakase, sp nov

In liquido “YM” post dies 5 ad 17°C, cellulae

vegeta-tivae sphaericae vel ovoideae aut ogiveae, 2.0–4.0

2.0–6.0 m m, singulae, binae, in catenis aut in fasciculis,

propagantes formatione conidiorum stipitatorum et

gemmarum blasticarum Post unum mensem ad 17°C,

pellicula fragilis et sedimentum formantur Cultura in agaro “YM,” gilvus-flava, rugosa, non nitida et margine erosa Pseudomycelium formatur Ballistoconidia apic-ulata, 2.0–3.0
3.0–6.0 mm

Fermentatio nulla Glucosum, galactosum, L -sor-bosum (lente et exiguum), sucrosum, maltosum, cel-lobiosum, trehalosum, lactosum, melibiosum, raffi-nosum, melezitosum, amylum solubile, D-xylosum, L -arabinosum (lente), D-arabinosum (exiguum), D

-ribo-Table 3. Sequence similarities of ITS region among five Kockovaella isolates and related species a

aLower left triangle shows sequence similarities in ITS1 and upper right triangle shows them in ITS2.

JCM 10051 JCM 10164 JCM 10165 JCM 10166 JCM 9862

Table 4. DNA-DNA reassociation experiment among five Kockovaella isolates and related species.

% relative binding of DNA from

ND, not determined.

JCM 10051 VY

sum, L-rhamnosum (lente), galactitolum (lente et

ex-iguum), D-mannitolum (lente), D-glucitolum (exiguum),

salicinum (lente et exiguum), glucuno-d -lactonum

(lente et exiguum), acidum 5-ketogluconicum (lente et

exiguum), acidum D-glucuronicum (lente), acidum D

-galacturonicum (lente), acidum succinicum (lente) et

acidum citricum (lente) assimilantur at non inulinum,

ethanolum, glycerolum, erythritolum, ribitolum, a

-methyl-D-glucosidum, acidum 2-ketogluconicum,

acidum DL-lacticum nec inositolum Ammonium

sulfa-tum et L-lysinum assimilantur at non kalium nitricum,

natrium nitrosum, cadaverinum nec ethylaminum

Maxima temperatura crescentiae: 31–32°C Ad

cres-centiam thiaminum necessarium est et niacinum

stim-ulare Materia amyloidea iodophila non formantur

Ureum hydrolysatur Diazonium caeruleum B:

Posi-tivum Proportio molaris guaninicytosini in acido

de-oxyribonucleico: 53.5 mol% per HPLC Systema

ubiquini: Q-10 Xylosum in cellulis presens

Holotypus: Isolatus ex folio Calophylli membrana-cei, Vietnam, JCM 10842/VTCC1 0180 (originaliter ut

VY-137) conservatur in collectionibus culturarum quas

“Japan Collection of Microorganisms,” Wako, Saitama

et “Vietnam Type Culture Collection,” Hanoi, Vietnam sustentat

Growth in YM broth: After 5 days at 17°C, the veg-etative cells are spherical to ovoidal with a few ogival ones and measure 2.0–4.0
2.0–6.0 mm They occur singly, in pairs or in groups, and reproduce by budding and stalked conidia (Fig 3) Stalked conidia are ob-served in both pellicle and sediment The conidia are separated at the distal end of the stalks from parent cells A sediment and a dull, wrinkled climbing pellicle are formed after one month at 17°C

Growth in YM agar: After one month at 17°C, the streak culture is light yellow, wrinkled, dull, and rough

(A)

netted, and it has an erose margin Stalked conidia

and ballistoconidia are produced

Dalmau plate culture on corn meal agar:

Pseudomycelium is formed after 2 weeks of incubation

at 17°C

Formation of ballistoconidia: Ballistoconidia are

formed abundantly on corn meal agar after 3 days

in-cubation at 17°C (Fig 3) They are symmetrical and

apiculate, measuring 2.0–3.0
3.0–6.0 mm

Fermentation: Absent

Assimilation of carbon compounds:

Fig 2. Primary structure of the ITS1 (A) and ITS2 (B) sequences among five Kockovaella isolates and related species

K_shim_10051, Kockovaella shimae JCM 10051; F_chin_10164, Fellomyces chinensis JCM 10164; F_lich_10165, F lichenicola JCM 10165; F_sich_10166, F sichuanensis JCM 10166; F_dist_9862, F distylii JCM 9862

(B)

T Type strain.

a, positive;

b Canete-Gibas et al (1998).

cHamamoto et al (1998).

Vitamin requirements and vitamin stimulation

Maximum growth temperature (°C)

Formation of ballistoconidia

L

-Sorbose Lactose Melibiose Raffinose

L

-Arabinose

D

-Arabinose

D

-Ribose

L

-Rhamnose Glycerol Erythritol Ribitol Galactitol

D

-Mannitol

D

-Glucitol Salicin

Glucono-d

-lactone

2-Ketogluconic acid 5-Ketogluconic acid

DL

-Lactic acid Succinic acid Citric acid Inositol

D

-Glucuronic acid

D

-Galacturonic acid Potassium nitrate Ethylamine hydrochloride Cadaverine dihydrochloride

Glycerol 

a -Methyl-D-glucoside 

Glucono-d -lactone  (latent and weak)

2-Ketogluconic acid 

5-Ketogluconic acid  (latent and weak)

D-Glucuronic acid  (slow)

D-Galacturonic acid  (slow)

Assimilation of nitrogen compounds:

Ethylamine hydrochloride 

L-Lysine hydrochloride 

Cadaverine dihydrochloride 

Maximum growth temperature: 31–32°C

Vitamins: Thiamine required and niacin stimulated

Production of starch-like substances: Negative

Diazonium blue B color reaction: Positive

Urease: Positive

Liquefaction of gelatin: Negative

Acid production on chalk agar: Negative

GC content of nuclear DNA: 53.5 mol% (by

HPLC)

Ubiquinone system: Q-10

Xylose in the cells: Present

Strain examined: Strain VY-137 was isolated by

Dao Thi Luong in February 1999 from a leaf of Calo-phyllum membranaceum Gard (Guttiferae), collected

by Dao Thi Luong and Takashi Nakase at Cuc Phuong National Forest of Ninh Binh, Vietnam This strain has been deposited in Japan Collection of Microorganisms (JCM), RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan, and at the Vietnam Type Culture Collection (VTCC), Centre of Applied Mi-crobiology, University of Science, Vietnam National University, Hanoi, Vietnam, with the accession num-bers JCM 10842 and VTCC1 0180, respectively

Kockovaella cucphuongensis Luong, Takashima, Ty,

Dung & Nakase, sp nov

In liquido “YM” post dies 5 ad 17°C, cellulae vegeta-tivae sphaericae vel ovoideae aut ogiveae, 3.0–5.0
3.0–7.0 m m, singulae, binae, in catenis aut in fasciculis, propagantes formatione conidiorum stipitatorum et gemmarum blasticarum Post unum mensem ad 17°C, pellicula fragilis et sedimentum formantur Cultura in agaro “YM,” gilvus-flava, rugosa, non nitida et margine erosa Pseudomycelium formatur Ballistoconidia apic-ulata, 2.5–3.5
2.5–7.0 mm

Fermentatio nulla Glucosum, galactosum, L -sor-bosum (exiguum), sucrosum, maltosum, cellobiosum, trehalosum, lactosum, melibiosum (lente), raffinosum (lente), melezitosum, amylum solubile, D-xylosum, L -arabinosum (lente), D-arabinosum (lente), D-ribosum,

L-rhamnosum (lente), erythritolum (lente et exiguum), ribitolum (lente), galactitolum (lente), D-mannitolum (lente), D-glucitolum (lente), salicinum (exiguum), glu-cono-d -lactonum (lente), acidum 2-ketogluconicum (lente), acidum 5-ketogluconicum (exiguum), acidum

D-glucuronicum, acidum D-galacturonicum, acidum succinicum (lente) et acidum citricum (lente), assimilantur at non inulinum, ethanolum, glycerolum, a -methyl-D-glucosidum, acidum DL-lacticum nec inosi-tolum Ammonium sulfatum et L-lysinum assimilantur

at non kalium nitricum, natrium nitrosum, cadaverinum nec ethylaminum Maxima temperatura crescentiae: 29–30°C Ad crescentiam thiaminum necessarium est Materia amyloidea iodophila non formantur Ureum hy-drolysatur Diazonium caeruleum B: Positivum Propor-tio molaris guaninicytosini in acido deoxyribonucle-ico: 53.7 mol% per HPLC Systema ubiquini: Q-10

Xy-Fig 3. Kockovaella calophylli JCM 10842.

(A) Vegetative cells grown in YM broth for 5 days at 17°C (B)

Ballistoconidia produced on corn meal agar after 3 days at

17°C Scale bars indicate 10 m m.

losum in cellulis presens.

Holotypus: Isolatus ex folio Smilacis glabrae,

Viet-nam, JCM 10840/VTCC1 0177 (originaliter ut VY-125)

conservatur in collectionibus culturarum quas “Japan

Collection of Microorganisms,” Wako, Saitama et

“Viet-nam Type Culture Collection,” Hanoi, Viet“Viet-nam

susten-tat

Growth in YM broth: After 5 days at 17°C, the

veg-etative cells are spherical to ovoidal with a few ogival

ones and measure 3.0–5.0
3.0–7.0 mm They occur

singly, in pairs or in groups, and reproduce by budding

and stalked conidia (Fig 4) Stalked conidia are

ob-served in both pellicle and sediment The conidia are

separated at the distal end of the stalks from parent

cells A sediment and a dull, wrinkled climbing pellicle

are formed after one month at 17°C

Growth in YM agar: After one month at 17°C, the

streak culture is light yellow, wrinkled, dull, and has an

erose margin

Dalmau plate culture on corn meal agar:

Pseudomycelium is formed after 2 weeks of incubation

at 17°C

Formation of ballistoconidia: Ballistoconidia are

formed abundantly on corn meal agar after 3 days

in-cubation at 17°C (Fig 4) They are symmetrical and

apiculate, measuring 2.5–3.5
2.5–7.0 mm

Fermentation: Absent

Assimilation of carbon compounds:

a -Methyl-D-glucoside 

Glucono-d -lactone  (latent) 2-Ketogluconic acid  (latent) 5-Ketogluconic acid  (weak)

D-Galacturonic acid 

Assimilation of nitrogen compounds:

Fig 4. Kockovaella cucphuongensis.

(A) Vegetative cells grown in YM broth for 5 days at 17°C of JCM 10840 (B) Ballistoconidia produced on corn meal agar after 3 days at 17°C of JCM 10840 (C) Vegetative cells grown

in YM broth for 5 days at 17°C of JCM 10839 (D) Ballistoconi-dia produced on corn meal agar after 3 days at 17°C of JCM

10839 Scale bars indicate 10 m m.