First Report of Needle Blight on Blue Pine (Pinus wallichiana) and Aleppo Pine (P. halepensis), Caused by Lophodermium macci, from Asia
Farooq A. Ahanger,
Gh. Hassan Dar,
Muzafar A. Beig
Blue pine (Pinus wallichiana), the dominant species of pine widely grown in the Himalayan region including Jammu and Kashmir State, has been found affected by needle cast disease. Recently during forest survey for needle blight diseases conducted in the year 2010 a new fungal pathogen, Lophodermium macci Sokolski and Berube, was identified for the first time from Asia with the help of morphological characterization and ITS-1 DNA sequencing technique. A free hand section in lactic acid fuchsin was used for morphological characterization. For molecular identification DNA was isolated from hysterothecia formed on the infected needles. The fungus, previously recorded on Pinus strobus, has been noticed for the first time on Blue pine (Pinus wallichiana) and Aleppo pine (P. halepensis). The pathogen L. macci is closely (98%) related to L. pini-excelsae and L. nitens. The conidiomata of L. macci which have not previously been documented were observed for the first time. The conidiomata were subepidermal dark brown to black in color. Black zone lines were only present when adjacent to other Lophodermium species. Since Lophodermium macci Sokolski and Berube has not so far been reported from any of the Asian countries so this forms the first report of this fungus from Asia. Further, the presence of L. macci on Pinus wallichiana and P. halepensis are new host records worldwide.
July 10, 2011; Accepted: September 28, 2011;
Published: December 12, 2011
Blue pine, Pinus wallichiana (Jack.), belongs to the genus Pinus
and family Coniferae. It is an evergreen arbor of monotypic genus in family
Pinaceae widely growing in Jammu and Kashmir State. The tree is abundantly found
in moist and dry temperate forest types of Western and Central Himalayas from
Kashmir to Bhutan at an altitudinal zonation of 1700-3700 mamsl (Lazarev
et al., 2007).
Blue pine (Pinus wallichiana) and Allepo pine (Pinus halepensis),
are often damaged by insects and pathogens (Huang et
al., 2008). Among the diseases, Lophodermium needle blight is a serious
disease incited by various Lophodermium species (Hou
et al., 2006, 2009). At first, the symptoms
appear as bar-like circular or sub-rounded yellowish-brown spots on needles.
Then the needles turn yellow and wither, even may fall off in autumn. Extensive
needle drop not only reduces the ornamental value of conifers, but also influences
the tree growth (Xiaoming et al., 2010). Among
all the needle blight diseases of pine Lophodermium needle blight is considered
as a major constraint in its successful regeneration (Johnston,
2001; Stenstrom and Ihrmark, 2005; Luo
et al., 2010). Initially the symptoms appear on needles as light
green to yellow spots which later turn reddish brown surrounded by yellow margins
(Xiaoming et al., 2010; Lilja
et al., 2010). The affected needles turn straw coloured and fall
off prematurely in autumn (Diwani and Millar, 1987;
Barnard and Ash, 1998). Extensive needle drop not only
reduces the ornamental value of conifers, but also influences the tree growth
(Douce et al., 2002; Hanso and
Lophodermium Chevall. is a large and complex genus in the family Rhytismataceae
and includes more than 250 species of foliar inhabiting fungi (Johnston,
2001). Lophodermium species have a wide host spectrum, including
families of gymnosperm and angiosperm (Luo et al.,
2010). Genus Lophodermium has received much attention by forest pathologists
because some species are important pathogens of conifers causing needle blight
diseases (Dabker, 1932; Minter, 1981).
Worldwide more than 25 species of Lophodermia have been reported on conifers
(Minter, 1981, 1995; Sokolski
et al., 2004; Hou et al., 2006, 2009).
From Asia about 15 Lophodermium species have been reported (Sharma
and Sharma, 1983; Bilgrami et al., 1991; Ojha
et al., 2004). After consulting the related iteratures ofconifer-inhabiting
Lophodermium spp. (Hou and Wang, 1995; Johnston,
2001; Johnston et al., 2003; Sokolski
et al., 2004; Hou et al., 2006, 2009;
Xiaoming et al., 2010) and comparing the pathogen
on P. wallichiana with L. pinastri, L. pini-execlsae and
L. nitens, it was found that there were great variations between the
three species. The pathogen on P. wallichiana and P. halepensis,
therefore, is identified as a new species of Lophodermium here. In the
present study, a new species of Lophodermium isolated from the conifer
needles, causing needle blight disease in Pinus wallichiana and P.
halepensis is reported on the basis of conventional and molecular identification
MATERIALS AND METHODS
Study area: In April 2010, Blue pine (Pinus wallichiana) and Allepo pine (Pinus halepensis) needles bearing typical needle cast (blight) symptoms were collected from conifer forests of Baramulla, Srinagar and Anantnag districts in Jammu and Kashmir State, India. The spores from ascomata formed on fallen needles of blue pine were used for the isolation of causal pathogen.
Morphological studies: For various morphological studies, the sections
of different thickness of ascoma were made in water containing 0.1% (w/v) cotton
blue in lactic acid as per Hou et al. (2009).
The fungus was identified on the basis of cultural and morphological characters
and characterized to species level using the keys of Minter
(1981). The pathogenecity was established on two year old Blue pine and
Allepo pine seedlings by using the perfect state spore of the fungus. The diseased
needle specimens were preserved in the Mycology and Forest Pathology Laboratory,
Division of Plant Pathology, SKUAST-K, Shalimar, Srinagar (J and K). For observing
the outlines of ascomata and conidiomata, vertical sections were mounted in
lactic acid or cotton blue with pretreatment in water. Gelatinous sheaths surrounding
ascospores and paraphyses were observed in water or cotton blue in lactic acid.
Ascospore contents were observed in water. Measurements for ascospores and paraphyses
were made using material mounted in 5% KOH from 30 ascospores, asci and paraphyses
for each specimen. The figures of external shapes and internal structures of
fruiting bodies were drawn using the microscopic painting device (Magnus Live
USB 2.0 camera; Magnus Pro 4).
Molecular methods: The molecular sequencing of the pathogen was carried
out followed by Hou et al. (2009) Total genomic
DNA was extracted from ascomata or mycelium as per Hou et
al. (2009). New sequence for ITS rDNA region was obtained for species
of Lophodermium macci. The primers ITS1 were chosen and PCR was performed
in 25 μL reaction (White et al., 1990; Hou
et al., 2009). The PCR products were sent to Capital Normal University
Xisanthuanbeilu, Beijing China for purifying, sequencing and editing.
During the survey for needle cast/blight disease of blue pine in selected areas
of Kashmir, several pine needles were noticed to have ascocarps without any
transverse black zone lines, a characteristic symptom of needle cast caused
by Lophodermium macci. The ascoma were mostly found on abaxial surface
and rarely on adaxial surface of the needles. The anamorph (Fig.
1d, e) of the isolated pathogen was found to be Leptrostroma
species. The identification of L. macci was subsequently confirmed
by molecular sequencing technique using ITS-1 primer (Fig. 2)
at Capital Normal University Xisanthuanbeilu, Beijing China. Comparison of morphological
and molecular data with other species and ribosomal DNA sequence of isolates
showed high level of genetic similarity (98% identity) for the internal transcribed
spacer region of L. pini-excelsae.
Symptomatology of disease: The disease on needles initially appeared
as yellowish spots which later turned brown with yellowish halos. The spots
on needle later coalesced to give blighted appearance (Fig. 1a)
and ultimately resulted in premature defoliation. The stereoscopic examination
of blighted needles showed the presence of small pycnidia (spermatia) from June
to July (Fig. 1d). The presence of conidiomata in this species
was a conspicuous observation which has not so far been reported elsewhere on
pines. Conidia were 8.34x1.60 μm in size (range, length 6.42-10.72 μm
and width 1.09-1.93 μm) (Fig. 1e). The mature hysterothecia
of fungus, singly or in chains with prominent slit in centre and surrounded
by grayish-shiny perimeter line, were abundantly found on diseased needles from
July to September (Fig. 1f). In field, the ascospores were
discharged from early July to early October, with vigorous dispersal from late
July to early September.
Cultural-morphological characterization: The ascoma on Pinus wallichiana
and P. halepensis needles were initially sub-epidermal but a t maturity
||(a) Blue pine needle showing needle cast disease symptoms;
(b) culture of Lophodermium macci (c) pycnidial formation on culture
plate; (d) Pycnidia of L. macci; (e) conidia, (f) hyserothecia and
(g) acsi and paraphysis
|| Primer sequence used to amplify partial Lophodermium macci
r-DNA ITS region
When picked these were observed to be deep-seated inside the plant tissue. Ascomata on both the sides of needles (more than 80% on adaxial side) were usually scattered and occasionally confluent. In surface view, ascomata were 500-900x300-500 μm, and clypeus 400-650x150-300 μm in size. The ascoma were mostly rounded with elliptical ends and occasionally acute or slightly irregular. Ascoma were shiny, black in centre for 1/3-1/2 (-2/3) surface area surrounded by grey or grey-brown margin with a conspicuous black perimeter line. The perimeter lining was moderately raised on the surface of the needle. The ascoma were opened by a single longitudinal slit. The lips of ascoma were inconspicuous under dissecting microscope but visible in thin sections under compound microscope. The lips were split extending 1/22/3rd length of ascoma.
The fungus was easy to isolate on malt extract agar (MEA), however good growth was observed on potato dextrose agar (PDA) after transfer. The colonies had irregular margins. The mycleium was creamish white with sparse growth and grew slowly. After 3-4 week, creamish buff pycnidial initials were observed in the form of concentric rings. The pycnidia later on turned black.
Paraphyses were rare, and when present were septate having hooked tips and
measured 70-110x2 μm. Asci were 70-110x7-8 μm in size (Fig.
1g) and ascospores 55-80x2 μm, filiform, arranged in fascicles, sometimes
helically coiled, slightly tapering toward base, hyaline and aseptate (Table
1). The discharged ascospores were covered with a gelatinous sheath of 2-3
μm thickness. Leptrostroma/conidiomata stage was present, mostly on adaxial
side of needles, scattered to crowded, sometimes coalescing. In surface view
conidiomata measured 500-750 (640)x350-630 (490) μm and conidia 8.34x1.60
μm in size (range, length 6.42-10.72 μm and width 1.09-1.93 μm).
Zone lines were absent.
The fungus was easy to isolate on malt extract agar, however good growth was observed on potato dextrose agar after transfer. The colonies had irregular margins.
||Comparative morphological characteristics of L. macci and
The mycelium was whitish farinaceous to creamish with sparse growth and grew slowly (Fig. 1b). After 3-4 weeks, the creamish buff pycnidial initials were observed in the form of concentric rings (Fig. 1c). The pycnidia later on turned black.
Ecology: Lophodermium macci has been observed on both intact as well as on fallen needles (over-wintered in litter) of P. wallichiana (five-needle) and P. halepensis (two-needles) pines in Kashmir conifer forests.
Habitat: Collected from fallen needles in litter and on host (P. wallichiana and P. halepensis).
Known distribution: Canada and USA on Pinus strobes.
Molecular characterization of pathogen: The molecular sequencing of
ITS region of DNA from the isolated fungus, using ITS-1 primer (Table
1) has been assigned accession number 37441 by the NCBI and sequence published
under the same accession number. On the basis of morphological and molecular
sequencing the pathogen has been identified as Lophodermium macci Sokolski
and Berube, hitherto unrecorded from Asian continent. The taxon has recently
been reported only from Canada and USA on Pinus strobus (Sokolski
et al., 2004) so this is the first host record of Lophodermium
macci on Pinus wallichiana and Pinus halepensis.
The Lophodermium macci Sokolski and Berube is an ascomycetous fungus
which has so far been reported from USA and Canada only that too on only one
pine species Pinus strobus, thus the present report is a new record from
Asian continent. In Jammu and Kashmir the fungus causes needle blight disease
in two pine species Pinus wallichiana (five-needle) and P. halepensis
(two-needle) which is the new host-fungus interaction from the world. Only three
species of Lophodermium have been reported from Jammu and Kashmir State
(India) viz., L. pinastri and L. pini-excelsae on Pinus wallichiana
and L. picea on Abies pindrow (Bagchee and
Singh, 1960; Sharma and Sharma, 1983). A study of
freehand sections of ascocarps mounted on lactic acid fuchsin (Hou
et al., 2009) and use of identification keys of (Minter,
1981) led to the identification of pathogen as Lophodermium macci
Sokolski and Berube.
Lophodermium macci belongs to the species complex whose members often
are difficult to differentiate (Sokolski et al.,
2004). Lophodermium macci is morphologically similar to L. pinastri
and L. pini-excelsae However, they are different in many aspects:
L. macci is characterized by the smaller proportions of the central black
area in the external appearance and the central longitudinal split in the total
length of ascoma, the covering stroma connecting to the basal stroma, and paraphysis
tips which are gradually swollen or irregularly branched even suddenly swelling
to globules. Besides, the anamorph of the present species is different from
L. pinastri and L. pini-excelsae. In L. macci, conidiomata
are flask to pear shaped and opened by a round ostiole on the top; its conidia
are much longer (8.34x1.60 μm) and zone lines are absent. Whereas L.
pinastri has the covering stroma not extending to the basal stroma, simple
paraphysis tips and longer asci and ascospores (110-155 μm and 70-140 μm,
respectively (Dabker, 1932; Minter,
1981), and elliptical to elongated elliptical conidiomata opened by a lateral
split, shorter and thinner conidia 4.5-6.25x1.0 μm (Minter,
1981) and the abundant zone lines (Minter et al.,
1978; Minter, 1980; Lin and
Tang, 1988; Johnston, 2001). Comparisons of ascomata
of L. macci with those of L. pinastri, L. staleyi and L. pini-excelsae
were described by Minter (1981). The similarities
among these species are the black clypeus with grey margin, a black perimeter
line and subepidermal nature. The most useful characteristic for distinguishing
L. macci from these species is the manner in which the ascocarp is embedded
in the needle (Table 1). This is the principal criterion separating
L. pinastri from L. pini-excelsae used by Minter
et al. (1978) and Cannon and Minter (1986).
In this case the difference is in the depth of embedded ascocarps. Lophodermium
macci displaces two or three epidermal cells (rarely four) of the host on
each side of the clypeus, while L. pini excelsae displaces more than
four, usually five. The number of epidermal cells left on the bottom of the
ascocarp is five and more for L. pini-excelsae and eight and more for
L. macci. The species of Lophodermium on conifers have high
host specificity at their generic level of the hosts (Hou
et al., 2009). Therefore, combining the morphology, host specificity
and molecular analyses, the species of Lophodermium on P. wallichiana
and P. halepensis is proposed as a new record from Asian continent.
Lophodermium macci, characterized by subepidermal black hysterothecia and surrounded by sharply defined greyish perimeter line, is a new report from Asia and its presence on Pinus wallichiana and P. halepensis are new host records worldwide. The conidiomata which have not previously been documented were observed for the first time. The conidiomata were subepidermal dark brown to black in colour. Black zone lines were only present when adjacent to other Lophodermium species.
We are grateful to Dr Cheng-Lin Hou, College of Life Science, Capital Normal University, Xisanthuanbeilu, Beijing 100048, Peoples Republic of China for the help rendered in molecular identification and sequencing of fungus and also for providing valuable suggestions from time to time in this study.
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