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Submitted 21 October 2014, Accepted 28 February 2015, Published online 11 March 2015
Corresponding Author: Swapnil Kajale – e-mail – swapnilckajale@gmail.com 133
Leptoxyphium kurandae - New record of insect gut associated sooty
mould fungus from India
Kajale SC, Sonawane MS, Sharma R and Shouche YS
Microbial Culture Collection, National Centre for Cell Science, NCCS Complex, Ganeshkhind, Pune–411 007
(Maharashtra), India
Kajale SC, Sonawane MS, Sharma R, Shouche YS 2015 – Leptoxyphium kurandae - New record of
insect gut associated sooty mould fungus from India. Mycosphere 6(2), 133–138, Doi
10.5943/mycosphere/6/2/2
Abstract
During a survey to study insect associated fungi, a sooty mould fungus, Leptoxyphium
kurandae found in a gut of insect (Dusky Cotton bug) from Western Ghats, Junnar, India was
isolated. It is characterized by elongated synnemata consisting of hyphae with bulbous base and at
apex an open terminal funnel shaped conidiogenous zone. The hyphae composed of cylindrical
cells, constricted at septa and covered with thick mucilaginous layer. Based on morphological
characters and sequence comparison of the internal transcribed spacer (ITS) of rDNA and large
subunit of ribosome (LSU), the fungus was identified as Leptoxyphium kurandae, a species not
previously known from India.
Key words – Dusky cotton bug – ITS – LSU – Synnemata
Introduction
Western Ghats are major diversity hotspot for both flora & fauna. Plant diversification has
been implicated as a major factor affecting the diversity of bugs, beetles & other insect groups
(Farrell 1998). Insect associated fungi & also their crucial role in nutritional requirement for insect
had remained under study (Suh et al. 2005). Due to less studies on insect gut associated fungi,
survey was conducted in Western Ghats to isolate fungi. Species of Leptoxyphium causes a kind of
sooty mould & belongs to the family Capnodiaceae. They are found to be restricted to the glandular
trichomes of leaves. Leptoxyphium occurs worldwide in form of sooty mould & frequently in pure
colonies (Hughes 1976). Though Leptoxyphium is plant pathogenic fungi, the association is not
observed in insects so far. Recently two new species, Leptoxyphium madagascariense
(Cheewangkoon et al. 2009) on leaves of Eucalyptus camaldulensis & L. kurandae (Crous et al.
2011) on leaves of Eucalyptus sp. were described. Literature survey of compilation of Indian fungi
shows that so far six species from genus Leptoxyphium are recorded earlier from India which
includes L. axillatum (Hughes 1976) on leaves of Albizia lebbeck (L.); L. bahiense (Batista 1963)
on leaves of Gossypium barbadense (L.), G. arboretum (L.), G. hirsutum (L.); L. fumago
(Srivastava 1982) on leaves of Kydia pinnata (L.), Ilex diopyrena (L.), Alnus nepalensis (L.),
Mangifera indica (L.), Quercus leucotrichophora (L.), Coix lycrima-jobi (L.), Rhododendron
arboretum (L.); L. graminum Patouillard (Spegazzini 1918) on Citrus sp.; L. longispora (Jain &
Mehta 2004) on leaves of Hibiscus rosa-sinensis (L.) & Leptoxyphium sp. (Spegazzini 1918) on
Mycosphere 6 (2): 133–138(2015) ISSN 2077 7019
www.mycosphere.org Article Mycosphere
Copyright © 2015 Online Edition
Doi 10.5943/mycosphere/6/2/2
134
Crescentia pinnata (L.); L. zeae (Mukerji & Khanna 1983) on leaves of Zea mays (L.) (Mukerji et
al 1983, Bilgrami et al 1991, Jain et al. 2004, Jamaluddin et al. 2004). However, L. kurandae is not
reported from India. We obtained a culture of Leptoxyphium kurandae from the gut of Dusky
Cotton bug (Lygaeidae) which feeds on leaves (Vennila 2007).
Materials & Methods
Isolates and morphology
Bug was collected from leaves directly and transported alive to the laboratory. It was
surface disinfected with 95% ethanol and rinsed in 0.7% saline solution before removal of the gut
and dissected out aseptically. Gut was suspended in 0.7% saline, homogenized by using pestle
which was used as stock. Microbial load was reduced using serial dilution by 10 fold diluting stock
solution. One unit of stock was removed and added in next tube containing 9 unit of 0.7% saline
called 1st dilution ie. 10-1 and dilutions are made up to 10-4 which were then spread on to the 2%
PDA plates supplemented by antibiotics. Isolated fungal colonies were purified on 2% PDA plates.
Out of these, one was selected for further study on the basis of molecular data. A herbarium
specimen was deposited in the Ajrekar Mycological Herbarium (AMH), India (AMH 9616); a
culture was deposited at Microbial Culture Collection (MCC), India (MCC 1085) and in
Centraalbureau voor Schimmelcultures (CBS), Netherlands (CBS 135836).
For morphotaxonomic studies and photomicrographs, Olympus BX53 microscope
(Olympus, Japan) was used fitted with ProgRes C5 camera (Jenoptik, USA). For microscopic
examination, fungal colony was mounted on glass slides with lactic acid cotton blue (Hi-Media).
Measurements of all taxonomically relevant parameters were made by Jenoptik, USA. Colony
characteristics in culture were studied on 5 different media: 2% malt extract agar (MEA), potato
dextrose agar (PDA), 2% oat meal agar (OA), potato carrot agar (PCA) and Czapek dox agar
(CDA). To test optimum temperature for growth, culture plates PDA, CDA, MEA, PCA, 2% OA
were incubated at 5 – 45 ± 2ºC temperatures range.
DNA extraction, PCR and sequencing
Genomic DNA was isolated from fungal mycelium grown on PDA, using QIAamp® DNA
Mini Kit (Qiagen, Inc., Valencia CA) as per manufacturer’s instructions and quantified by
NanoDrop spectrophotometer (ND-1000, Thermo scientific, USA). Two universal primer pairs
ITS1-ITS4 (White et al. 1990) and LR7-5.8S (Vilgalys & Hester 1990) were used separately for
amplification of the internal transcribed spacer (ITS1-5.8S-ITS2) region and D1/D2 region of the
large subunit (LSU) rRNA gene respectively by using PE 9700 thermocycler (PE Applied
Biosystems, Singapore). The PCR was carried out by standard techniques with the following
cycling program: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 1
min, annealing at 55°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 10
min and hold on 20°C. The purified double-stranded PCR product was used as templates for
sequencing with an ABI 3730 xl DNA sequencer using the Big Dye Terminator cycle sequencing
kit (Applied Biosystems, Inc., Foster City, CA). The sequence homology was carried by using
BLASTn search to compare it with available sequences of Leptoxyphium in GenBank database
(Zhang et al. 2000).
Leptoxyphium kurandae Crous & R.G. Shivas, Persoonia, 26: 145, 2011 Figs 1–2
MycoBank MB560176
Optimum temperature for growth was 20–25°C. Mycelium consisting of grey-brown hyphae
with 3.6–9.1 μm diam, septate, branched, constricted at septa, forming hyphal ropes, thick-walled,
smooth and frequently encased in mucoid sheath. Conidiomata (Fig. 2E) synnematous, separate or
in clusters of 2–3, erect, straight to slightly flexuous; bulbous base brown 25.1–39.5 × 25.6–28.1
μm ; (
x
= 32.7 × 26.47 µm, n = 10), cylindrical part dark olivaceous brown 23.3–67.7 × 11.8–23.3
μm ; (
x
= 44.8 × 15.5 µm, n = 10), and after 3 months (Fig. 2D) 518.1–1278 × 9.57–12.83 μm ; (
x
135
= 840.2 ×13.3 µm, n = 10), hyphal apex 22.3–65.7 × 19–68.9 μm ; (
x
= 44.2 × 38.3 µm, n = 10),
Conidiophores (Fig. 1A-C) subcylindrical to subulate, 0–2-septate, 3.5–29.6 × 1.0–3.3 μm ; (
x
=
15.8 × 1.8 µm, n = 15), tightly aggregated in apical part of synnemata. Conidiogenous cells
intercalary, terminal, phialidic, 1.4–5.1 × 0.5–2.1 μm ; (
x
= 2.4 × 1.0 µm, n = 10), tapering, with
parallel to wall and visible collarette. Conidia (Fig. 1D) broadly ellipsoid with rounded ends, 0-1
septate, eguttulate, hyaline, smooth, 4.1–11.7 × 1.3–3.9 μm ; (
x
= 6.8 × 2.2 µm, n = 50),
aggregating at apex of synnemata in drop of liquid (Fig. 1E).
Sexual stage – Not known
Fig. 1 – Leptoxyphium kurandae MCC 1085. A: apical structure; B-C: conidiophore &
conidiogenous cells; D: conidia; E: conidia produced in drop of liquid at top of synnemata. Bars
A-D 20 µm; E 100 µm.
Fig. 2 – Developmental stages of formation of synnematous conidiophores structure of L.
kurandae MCC 1085 in artificial culture media. Bars A-C, E 20 µm; D 100 µm. A: Cells
differentiation; B: rearrangement of cells; C: formation of synnematous conidiophore structure; D:
Complete developed synnematous structure; E: Phase contrast image of synnematous
conidiophores with hyphal apices
136
Culture characteristics - (25°C in dark, after 2 wks): Colonies were slow growing and
spreading, moderate aerial mycelium and even margins, reaching 28 mm diam after 2 wk; on malt
extract agar surface olivaceous grey VI’’’’’i (121), outer region isabelline III’’i (65), and pale hazel
IV’’’i (88) in reverse; on oatmeal agar surface olivaceous grey VI’’’’’i (121); on potato-dextrose
agar surface dark greenish olivaceous IV’’’i (90), grey olivaceous V’’’’i (107) in outer region and
reverse (Rayner 1970).
Material examined – India, Pune (Western Ghats), Maharashtra, from gut of Dusky Cotton
Bug, 10 January 2013, Swapnil Kajale (Holotype, AMH 9616; culture, MCC 1085, CBS 135836).
Gene sequences: KF826942 (ITS), KF826943 (LSU)
Notes – The genus Leptoxyphium Speg. typified by Leptoxyphium graminum (Pat.) Speg.
mainly characterized by the synnemata arising from helically twisting hyphae or ropes of repent
hyphae with terminal conidiogenous zone. Hyphae composed of cylindrical cells & mucilaginous
hyphal outer walls (Hughes 1976). This genus has been expanded to include eighteen legitimate
species according to mycobank.
Discussion
Based on BLASTn search of NCBI’s GenBank nucleotide database, the closest hits using
the ITS & LSU sequences of L. kurandae MCC 1085 are showed in Table 1. The sequences of ITS
& LSU domain confirms the closest species in terms of pairwise sequence similarity (using
BLASTn search tool) as L. kurandae (99% similarity), whereas the L. madagascariense with 98%
similar.
Table 1- NCBI-BLAST hits (ITS & LSU) against Leptoxyphium kurandae MCC 1085
ITS
Similarity (%)
LSU
Similarity (%)
Leptoxyphium kurandae (JF951150)
99
Leptoxyphium kurandae
(JF951170)
99
Leptoxyphium sp. TMS-
2011(HQ631026)
99
Microxyphium citri
(AY004337)
99
Leptoxyphium madagascariense
(GQ303277)
98
Leptoxyphium fumago
(GU214430)
99
Polychaeton citri (GU214649)
91
Leptoxyphium madagascariense
(GQ303308)
98
Capnodium coffeae
(GU214400)
98
There is variation in morphological characters viz. size of hyphal apex, cylindrical part,
conidiogenous cells and also branched synnematous structures showing slight variation in
morphological features from L. kurandae. Also the present species is very much different than L.
madagascariense (Table 2), but rDNA sequence comparisons (Table 1) showed that our isolate is
indeed L. kurandae with 99% similarity. As observed morphologically after 3 months, successive
proliferated fructifications were seen of which a length of cylindrical part increased drastically high
as 518.1–1278 x 9.57–12.83 μm in pure culture. Also in the old culture multiple secondary
synnemata arise from single conidiophore of a conidiomata to form a branched structure (Fig. 2D).
No reports were found that insect gut is a habitat of Leptoxyphium other than the insect honeydew
(sugar rich liquid). The present fungus might have entered in the gut of bug through the leaves on
which it was feeding or living. The role of Leptoxyphium is not known in gut of insect whether
pathogenic or symbiotic so far but it might be harmful to insect because of some phenolic
compounds produced by Leptoxyphium (Singh et al. 2005). Sequencing of rDNA shows that our
isolate is Leptoxyphium kurandae, a species not previously recorded from insect gut. Moreover,
there is no previous record of L. kurandae from India. The isolate is reported for the first time from
gut of Dusky cotton bug which is a common pest on cotton and leaves (Vennila 2007). Therefore,
the present fungus is reported as new record from insect gut and also from India.
137
Table 2- Comparison of closely identical Leptoxyphium species.
Parameters
L. kurandae MCC
1085
L. kurandae CBS
129530
L. madagascariense CBS 120605
Conidia
4.1‒11.7 × 1.3‒3.9 μm
(4–)6–7(–9) × 2–3 μm
4.5–5 × 3–3.5 μm
Conidiophore
3.5‒29.6 × 1.0–3.3 μm
15–25 × 2–3 μm
-
Conidiogenous cells
1.4‒5.1 × 0.5‒2.1 μm
7–10 × 2–2.5 μm
≤1 × 2.8 μm
Bulbous base
25.1–39.5 × 25.6–28.1
μm
30–50 × 25–35 μm
-
Cylindrical part
23.3–67.7 × 11.8‒23.3
μm
60–100 × 12–15 μm
(200–)250(–300) × (8–)10–12(–15)
μm
Hyphal apex
22.3–65.7 × 19–68.9 μm
30–50 × 25–40 μm
35‒50 × 35‒60 μm
Acknowledgements
The authors thank Department of Biotechnology (DBT), Government of India & Microbial
Culture Collection Project (BT/PR10054/NDB/52/94/2007) for funding and Director, National
Centre for Cell Science, India for providing laboratory facilities.
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