Clonostachys indicus

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INTRODUCTION
The north western India is rich in mycobiota and many new
species of hyphomycetes have been reported from this region
(Adamčik et al., 2015; Prasher and Singh, 2015a, b; Prasher
and Verma, 2014, 2015a, b, c). As a part of ongoing efforts of
exploring and documenting fungal diversity an interesting
species of Clonostachys Corda was isolated from dead
branches of Ficus virens (Miq.) Domin collected in
Chandigarh. A critical morphological and molecular
examination of the specimens revealed it to be an undescribed
species of Clonostachys.
MATERIALS AND METHODS
Clonostachys indicus was isolated from the bark of dead
twigs of Ficus virens, collected from Department of Botany,
Panjab University, Chandigarh, India. Microscopic
descriptions were made from 10 days old culture grown on
MEA at 24°C. Mycelia were stained in 1% Congo red and
structures were studied microscopically and photographed
using a Matrix Transmission Microscope (VRS-2f). All the
measurements were made with the help of Pro MED software.
The line drawings were prepared with the help of Olympus 2
li microscope with a drawing tube attached to it.
Measurements are given as n n , (n ) n n n (n ), where n =
1 2 1 4 3 5 2 1
minimum value observed; n = maximum value observed; n =
2 3
arithmetical means; n = first quartile; n = third quartile
4 5
(Schroers, 2001). Ex-type culture is deposited in the
herbarium of Botany Department, Panjab University,
Chandigarh (PAN).
Isolation: Pure culture was isolated on Malt Extract Agar
(MEA) medium (Malt Extract 20g, Agar agar 20g, distilled
water to make 1000 ml) by hyphal tipping method after seven
days of incubation at 24°C in Petri-plate (Fig 1). Two sets of
pure cultures were prepared and maintained on the MEA
plates at ±4°C with periodic transfer.
DNA extraction and amplification: Fungal strain was
maintained on PDA slant. DNA was extracted from cultures
TM
grown on PDA plates for two weeks at 24°C using HiPurA SP
Fungal DNA mini kit (HiMedia) by following the manufacturer's
instructions. Fragments containing the ITS region was amplified
using primer pair ITS1/ITS4 (White et al., 1990).
DNA amplification was performed in a 25 μl reaction using 2
μl of template DNA (25 ng), 1U of Taq DNA polymerase i.e.
0.5 μl (Finnzyme Phusion™ High-Fidelity DNA Polymerase-
F-530S), 5 μl of 5x Phusion HF Buffer, 0.5 μl of 10 mM of
each dNTPs (Genei, Bangalore, India), 1 μl of 10 pmol
primer, 15 μl HO (Sterile Ultra Pure Water, Sigma) to make
2
up 25 μl. Amplification in an Eppendorf Mastercycler
Gradient 5331 AG used the following parameters: 5 min at
95°C; 30 cycles of 1 min at 95°C, 1 min at 56°C, and 1 min at
72°C; and final 7 min extension step at 72°C for ITS region
amplification. The PCR products were purified with an
Axygen PCR cleanup kit (Axygen Scientific, CA, USA) and
sequenced with the same primers using the BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
USA). The sequencing reactions were run on an ABI 3100
automated DNA sequence (Applied Biosystems, USA).
TAXONOMY
Clonostachys indicus Prasher I. B. and Chauhan R. sp. nov.
Figs. 2-4
MycoBank MB 821402
DIAGNOSIS: Conidial masses slimy dome-shaped.
KAVAKA 48(1):22-26(2017)
Clonostachys indicus sp. nov. from India
*
Indu Bhushan Prasher and Radha Chauhan
Department of Botany, Panjab University, Chandigarh-160014, India
*Post Graduate Government College for Girls, Sector- 42D, Chandigarh-160036.
*Corresponding author Email: radhachauhann@gmail.com
(Submitted in November, 2016; Accepted on June 20, 2017)
ABSTRACT
Clonostachys indicus sp. nov. is described and illustrated from pure culture, obtained from bark of twigs of Ficus virens (Miq.) Domin, collected
from Chandigarh, India. The new species was disguised by morphological and in vitro cultural characters from its related species. Additionally,
internal transcribed spacer (ITS) rDNA sequence analysis support this species as distinct within Clonostachys. A comprehensive table of
Clonostachys species possessing dimorphic conidiophores has been provided.
KEY WORDS: Hyphomycete, conidia, conidiophores, systematic, ITS
Fig. 1 Colony of Clonostachys indicus on Malt Extract Agar
after 10 days.
22

Co n idiop h ores d i morph i c. Pr i m ary co nidio p hores
verticillium like, mononematous. Secondary conidiophores
bi- to quarter verticillate. Conidia hyaline, minutely curved,
distally broadly rounded with a laterally displaced hilum,
ovoid to subglobose.
Etymology: The specific epithet 'indicus' has been named
after the country of origin i.e. India.
Conidial masses white, pale yellow or pale orange, normally
as watery to slimy dome-shaped masses, less frequently in
imbricate chains. Conidiophores dimorphic. Primary
conidiophores verticillium-like, formed throughout the
colony, dominating towards the margin, mononematous,
arising from the agar surface; stipe longer than penicillus, 30-
78 μm long, 2.1-3.6 μm wide at base; penicillus 43.6-
65.7(77.7) μm high; phialides cylindrical but slightly tapering
towards the tips, (18 20.9-23.7-25.9 (35.9) μm long, (2.1)
2.4-2.8-3.2 (3.9) μm wide at base, longer than in secondary
conidiophores; each producing a small, hyaline drop of
conidia that frequently collapses to form a single head on
several phialides of the same metula or of several side-
branches; collarette lacking. Secondary conidiophores bi- to
quarter-verticillate, densely aggregated, formed from strands
of aerial mycelium; branches either divergent at acute angles,
adpressed or short when arising laterally from the hyphal
Fig. 2 Clonostachys indicus (a,b) Primary conidiophores with
phialides (c,d) Stipe of conidiophore arising from
vege tat ive hyp hae (e) Con idi a fro m p rim ary
conidiophores. Scale bars: ad = 10 µm, e = 5 µm.
Fig. 3 Clonostachys indicus (a, b, d) Secondary conidiophores
with phialides diverging at acute angles (c) Typical
conidiophore lacking branches and with divergent
phialides (e) Conidia from secondary conidiophores.
Scale bars: ad = 10 µm, e = 5 µm.
Fig. 4 Clonostachys indicus (a,b) Primary conidiophores with
phialides (c,d) Secondary conidiophores with phialides
diverging at acute angles (e) Conidia from primary and
secondary conidiophores. Scale bars: ad = 10 µm, e = 5 µm.
Indu Bhushan Prasher and Radha Chauhan 23

strands; stipe up to 78.5 μm, 3.2 μm wide at base; penicillus
(37.3) 39.9-75.8 μm high; metullae 10.5-14.9 (24.4) μm long,
23 (3.8) μm wide at base; phialides in loose whorls of 35, (10)
14.7-16.4-17.8 (24.8) μm long, (1.9) 2.2-2.4-2.5 (3.3) μm
wide at base, straight to slightly curved, narrowly flask-
shaped, tapering in the upper part, without a visible collarette;
intercalary phialides not observed. Conidia hyaline, minutely
curved, distally broadly rounded, with a laterally displaced
hilum, ovoid to subglobose, conidia from primary
conidiophores slightly curved to almost ellipsoidal, (4.2) 5.0-
5.6-6.3 (7.4) × (2.1) 2.3-2.7-2.9 (3.6) μm from primary
conidiophore, (3.9) 4.2-4.6-5.0 (5.6) × (2) 2.9-3-3.2 (3.7) μm
from secondary conidiophore. Perithecia not observed.
Ex-type culture examined: INDIA. CHANDIGARH: Panjab
University Campus 30º45′N, 76º46′E, 355m. isolated from
dead twigs of Ficus virens, I. B. Prasher and Radha Chauhan,
08. 09. 2011. (PAN 34502).
Phylogenetic analysis: ITS sequence from Clonostachys
indicus was manually edited using Chromas Lite software
(www.technelysium.com.au). Sequence derived in this study
was deposited in GenBank (ITS: KT291441). The newly
generated ITS sequence of Clonostachys indicus and those
downloaded from GenBank based on the top-scoring match
(Table 1) were added to a dataset that was used for the
construction of phylogeny. For phylogenetic analysis, the
sequences were aligned using Clustal W together with the
homologous region of ITS of closely related genera and species.
For construction of phylogenetic tree (Fig. 5), the matrix was
analyzed using Neighbor-Joining method of Molecular
Evolutionary Genetics Analysis (MEGA) software v6.0.
(Tamura et al., 2013). The percentage of replicate trees in which
the associated taxa clustered together in the bootstrap test (1000
replicates) are shown next to the branches (Felsenstein, 1985).
The evolutionary distances were computed using the Kimura 2-
parameter method (Kimura, 1980). The ITS gene dataset
comprised of 17 taxa. The alignment comprised 443 characters,
gaps and missing data were eliminated.
DISCUSSION
The genus Clonostachys was established by Corda (1839) with
type species C. araucaria. The genus is characterized by
penicillate condiophores and imbricate conidia. Later on several
species have been described and transferred from other genera
like Gliocladium, Penicillium and Verticillium into Clonostachys
by different authors (Link, 1816; Hawksworth and Punithalingam
1975; Domsch et al., 1980; Gams, 1984; Samuels, 1988; Schroers
et al., 1999). The anamorphs of Bionectria are classified into the
genus Clonostachys (Schroers, 2001), which are characterized by
penicillate and dimorphic conidiophores in many cases. The
primary conidiophores are mononematous: verticillium like or
penicillate like forming mucoid conidial masses (Bainer, 1905,
1907). The secondary conidiophores generally form imbricate
conidia collapsing to form slimy conidial masses (mainly in
sporodochia). Domsch et al. (1980) observed various colours of
conidial masses such as white, pale orange or green.
Clonostachys indicus is morphologically more similar to C.
byssicola in having similar primary and secondary conidiophore
but differs from it in having smaller primary phialides [(18) 20.9-
23.7-25.9 (35.9) μm long, (2.1) 2.4-2.8-3.2 (3.9) μm wide at base
in C. indicus and (12.4) 22.6-26.2-29.2(48) μm long, (1.4) 1.8-2-
2.2 (2.8) μm wide at base in C. byssicola] and longer secondary
phialides [(10) 14.7-16.4-17.8 (24.8) μm long, (1.9) 2.2-2.4-2.5
(3.3) μm wide at base in C. indicus and (7.6) 10.8-13.8-15.4 (27.8)
μm long, (1.4) 1.8-2-2.2 (2.8) μm wide at base in C. byssicola].
Moreover, the primary conidia are larger i.e. (4.2) 5-5.6-6.3 (7.4)
× (2.1) 2.3-2.7-2.9 (3.6) μm in C. indicus as compared to C.
byssicola i.e. (3.2) 4.4-5.2-5.8 (10.8) × (1.8) 2.4-2.6-2.8 (4) μm
Clonostachys indicus unlike C. byssicola does not form
sporodochia in culture conditions. It differs significantly from
anamorphs of subgenus Astromata, Epiphloea and Myrothecium
in possessing dimorphic conidiophores (monomorphic
conidiophores in later anamorphs) and from anamorphs of
subgenus Zebrinella in lacking setae (prominent in anamorphs of
subgenus Zebrinella). It differs significantly from species under
subgenus Bionectria with respect to morphological details given
in Table 2 (Schroers, 2001).
Taxon name Origin Substratum Strain Sequence
Accession
No. of ITS
Bionectria apocyni USA, New
York
dead stem of Apocynum
cannabinum
CBS 130.87 AF210688
Bionectria aureofulvella New Zealand root of tree CBS 195.93 AF358226
Bionectria byssicola Uganda Alchorea branches CBS 914.97 AF358252
Bionectria capitata Japan bark CBS 218.93 AF358240
Bionectria oblongispora Japan bark of dying tree of
Orixa japonica
CBS 100285 AF358248
Bionectria
pseudochroleuca
French Guiana
bark
CBS 192.94 AF358238
Bionectria pseudostriata Indonesia
bark
CBS 119.87 AF358251
Bionectria
sporodochialis
U.S.A., Puerto
Rico
bark
CBS 101921 AF210685
Bionectria
zelandiaenovae
New Zealand
bark of ?Agathis australis
CBS 100979 AF358229
Clonostachys agrawalii India
decomposing buffalo
horn from animal house
floor sweepings
CBS 533.81 AF358241
Clonostachys
rhizophaga
Switzerland
culture contaminant
CBS 361.77 AF358228
Clonostachys
rogersoniana
Brazil soil CBS 582.89 AF210691
Clonostachys rosea - - BAFC1646 KF765504
Clonostachys rosea f.
catenulata
- - - HQ596905
Clonostachys rosea f.
rosea
- - T64D HM052816
Clonostachys solani f.
solani
Netherlands tuber of Solanum
tuberosum
CBS 228.74 AF358243
Clonostachys indicus India twig of Ficus virens IBP 2 KT291441
Fig. 5 Neighbour-Joining tree of Clonostachys indicus based
on ITS sequence.
Clonostachys indicus sp. nov. from India
24

Species Primary Conidiophore Secondary Conidiophore Conidia (μm)
Stipe (μm) Penicillus
(μm)
Phialide (μm) Stipe (μm) Penicillus
(μm)
Phialide (μm)
C. agrawalii 10–60 long or
more, 4 wide at
the base
- 18.8–32–42 long, 2.2–
2.6–3.0 wide at base
- - 7–18.2 long, 2.2–3 wide
at base
(3.8–) 4.2–4.6–4.8 (–5.8) ×
(2.2–) 2.4–2.6–2.9 (–3)
C. aureofulvella 30–190 long,
3–5 wide at the
base
30–100
high
(10.6–) 16.4–20–22.4
(–33.4) long, (1.2–) 1.8–
2–2.2 (–3.2) wide at
base
- - (8.4–) 11.4–13.4–15.2
(–21.2) long, (1.6–) 2–
2.4–2.6 (–3) wide at base
(3.6–) 4.8–5.8–7 (–9) ×
(1.8–) 2.4–2.8–3.2 (–3.8)
C. byssicola 10–100 long,
3–5 wide at
base
- (12.4–) 22.6–26.2–29.2
(–48) long, (1.4–) 1.8–
2–2.2 (–2.8) wide at
base
- - (7.6–) 10.8–13.8–15.4
(–27.8) long, (1.4–) 1.8–
2–2.2 (–2.8) wide at base
(3.2–) 4.4–5.2–5.8 (–10.8 )×
(1.8–) 2.4–2.6–2.8 (–4);
from sporodochia (3.2–
4.8×2.8–3.8)
C. capitata 100 long, 3.5
wide at base
- (18.6–) 28.4–33.8–40.2
(–46.6) long, (1.4–) 2–
2.2–2.4 (–2.6) wide at
base
- - (8.8–) 11.6–13.6–15 (–
24) long, (1.6–) 2–2.4–
2.6 (–3) wide at base
(4.6–) 6–6.8–7.2 (–12.4) ×
(2.2–) 2.8–3.2–3.4 (–4.2)
C. compactiuscula 40–250 long,
2.5–4.5 wide at
base
40–130
high
(17–) 22.2–28–31.2 (–
56) long, (1.8–) 2–2.4–
2.6 (–3) wide at base
100 long, to 7
wide at base
60 high
(5.2–) 8.4–8.8–11 (–
17.2) long, (1.2–)
1.8–2–
2.2 (–3) wide at base
(3.9–) 5.4–6.6–7.5 (–12.4) ×
(1.5–) 1.9–2.2–2.5 (–3.2)
C. kowhaii - -
-
40–100 long,
to 5 wide at
base
-
(13.6–) 21.8–26.2–28.8
(–42) long, (2–) 2.2–2.6–
2.8 (–3.2) wide at base
(4.4–) 7.6–10.6–13.2(–18.2)
× (2.8–) 3.4–4–4.6 (–5.8)
C. macrospora inconspicuous,
sometimes
absent
-
-
-
-
(7–) 11.2–15.4–18.6
(–24.0) long, (2–) 2.2–
2.4–2.6 (–3.2) wide at
base
(6.0–) 11.2–13–15 (–20.2) ×
(3.2–) 4.6–5–5.4 (–7)
C. oblongispora - -
-
-
-
(11.8–) 13.2–18.2–20.4
(–38),
(2.6–) 2.6–3–3.2
(–3.8) wide at base
(9–) 2.6–13.6–14 (–19.8) ×
(2.6–) 3.2–3.6–3.8 (–4.2)
C. pseudochro-
leuca
50–180 long,
2–5.5 wide at
the base
40–90
high
(13.6–) 17.4–20.8–22.8
(–34) long, (1.3–) 1.6–
1.8–2 (–2.4) wide at
base
-
-
(6.2–) 10.2–11.4–12.6
(–17.6) long, (1.3–)
1.8–
1.9–2 (–2.2) wide at base
(3.2–) 4–4.4–4.6 (–6.4) ×
(1.2–) 2.2–2.2–2.4 (–3)
C. pseudostriata 50–300 long,
3.5–6 wide at
base
25–80
high
(11.6–) 16.2–22.8–26.8
(–44.2) long, (1.6–)1.8–
2.2–2.4 (–3.2) wide at
base
-
-
(7–)
12.2–15.8–18.8
(–
30.4) long,(1.8–)
2.6–
2.8–3
(–3.2) wide at base
(3.6–) 5–5.6–6.2 (–8) × (2–)
2.6–3–3.2 (–3.8); from
primary conidiophores 13 ×
4
C. rhizophaga (10–) 40–100
long, 2.5–5
wide at base
30–100
high
(15.6–) 22–28.4–34.2
(–48.2) long, (2.2–) 2.6–
2.6–2.6 (–3.2) wide at
base
-
-
(5.8–) 12.4–14.6–17.2
(–25.2) long, (2.2–) 2.6–
2.6–2.6 (–3.2) wide at
base
(4.8–) 5.8–6.4–7 (–9) ×
(2.4–) 2.6–3–3.2 (–4.2)
C. rogersoniana 60–200 long,
3–5 wide at
base
40–150
high
(14.9–) 20.8–23.6–26.8
(–38) long, (2.2–) 2.4–
2.6–2.6 (–3.2) wide at
base
70–160 long,
5–7.5 wide at
base
50–100 and
higher,
100
diameter
at
widest point
(10.2–)
11.8–12.4–13.4
(–16.6) long, (1.6–) 2.4–
2.6–2.8(–3.2) wide at
base
(4.8–) 5.8–6.6–7.2 (–9.6) ×
(2.2–) 3–3.2–3.8 (–4.2)
C. rosea f.
catenulata
- -
(25–) 29–31–37 (–45)
long, (1.6-) 2–2.2–2.4 (–
3) wide at base
-
-
(8–) 10.4–12–14 (–18)
long, (2–) 2.2–2.2–2.4
(–2.8) wide at base
(4–) 4.8–5–5.4 (–6) × (2.2–)
2.4–2.6–2.8 (–3)
C. rosea f. rosea (25–)70–200
long, 3.5–5.5
wide at base
-
(16.6–) 22.8–27.8–31.2
(–46.6) long, (1.6–) 2.2–
2.4–2.6 (–3.4) wide at
base
60–110 long,
3.5–6.5 wide
at base
30–60 high,
16–50
diameter
at
widest
point
(5.6–) 10.6–12.4–14.4
(–19.6) long, (1.2–) 2.0–
2.4–2.6 (–3.2) wide at
base
(5.2–) 7.6–8.2–9.0 (–15.4) ×
(2.2–) 2.8–3.2–3.4 (–4.8);
from secondary
conidiophore (4.2–) 4.8–
5.2–5.6 (–6.6) ×(2–) 2.4–
2.8–3 (–3.4)
C. solani f. solani 60–240 long,
3–6.5 wide at
the base
20–100
high
(11.2–) 15.6–19–20.6
(–38.6) long, (1.6–) 2–
2.2–2.4 (–3) wide at
base
-
50–100 high,
70–150 wide
(9–) 11.6–14–15.2 (–25)
long, (1.8–) 2.2–2.4–2.6
(–3.4) wide at base
(5–) 6.4–7–7.4 (–10) long;
from secondary
conidiophores (3.8–) 4.4–
4.8–5 (–6.8) × (2–) 2.4–2.6–
2.8 (–3.8)
C. solani f.
nigrovirens
- -
(11–) 14–16.6–18.6(–
22.8)
long, (1.6–) 2–2.2–2.4
(–3) wide at base
(8–) 11.2–13.4–14.4 (–
23.4) long, (2.4–) 2.6–
2.8–3 (–3.2) wide
4.2–9 × 2.1–3.4; from
secondary conidiophores
(3.4–)
4.2–4.6–4.8 (–6.4) × (2.2–)
2.6–2.8–3 (–3.8)
C. sporodochialis - -
-
-
50–120 high,
50–100 wide
(13.4–)
18.8–21.6–24.6
(–35.4) long, (1.6–)
1.8–
2–2.2
(–2.6) wide at base
(3.2–) 4.4–4.8–5.4 (–6.8) ×
(1.6–) 2.0– 2.2–2.2 (–2.6)
C. verrucispora 50 long, 3 wide
at base
- (15.6–) 19–26.2–33(–
41.6) long, (1.8–) 2–
2.2–2.4
(–2.8) wide at base
- - (4–) 10.4–13.6–16.6 (–
22.6) long, (1.4–) 2.0–
2.4–2.8 (–3.2) wide at
base
(5.6–) 7.4–8.4–9.2 (–15.6) ×
(2.2–) 3–3.2–3.6 (–4.4)
C. zelandiaenovae 50 long, 4 wide
at base
- (21.2–) 27–32.6–34 (–
46.6) long, (2.2–) 2.6–
2.8–3.2 (–3.2) wide at
base
- - (4.8–) 11.4–13.6–16 (–
20.6) long, (1.6–) 2.2–
2.4–2.6 (–3.4) wide at
base
(4–) 5.2–6–6.4 (–13.2) ×
(2.4–) 2.8–3–3.2 (–4.2)
C. indicus 30.0–78.0 long,
2.1–3.6 wide at
base
43.6-65.7
(–77.7)
high
(18.0–) 20.9–23.7–25.9
(–35.9) long, (2.1–) 2.4–
2.8–3.2 (–3.9) wide at
base
Up to 78.5, 3.2
wide at base
(37.3–) 39.9–
75.8 high
(10.0–) 14.7–16.4–17.8
(–24.8) long, (1.9–) 2.2–
2.4–2.5 (–3.3) wide at
base
(4.2–) 5.0–5.6–6.3 (–7.4) ×
(2.1–) 2.3–2.7–2.9 (–3.6)
from primary conidiophore;
(3.9–) 4.2–4.6–5.0 (–5.6) ×
(2.0–) 2.9–3.0–3.2 (–3.7)
from secondary
conidiophore
Table 2 Comparative account of Clonostachys spp. (after Schroers, 2001).
Indu Bhushan Prasher and Radha Chauhan 25

ACKNOWLEDGMENTS
One of us (Radha Chauhan) acknowledges the Department of
Science and Technology, Govt. of India, for the financial
support in the form of fellowship under PURSE grant during
the course of the study and to UGC for grant-in-aid under
DRS-III programme. We are also thankful to the Chairperson,
Department of Botany, Panjab University, for providing the
facilities used during part of the experiments in this study.
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