endophytica Diaporthe sp. Colony of the endophytic fungi from C. calisaya on PDA 87
Figure 5.10 Morphological character of Fusarium oxysporum M16 on C. calisaya. a and b. 7 days culture on PDA, c. Chlamydospore. d. Macroconidia
and microconidia, d. Phialid. Bar a,b = 1 cm c,d = 20 μm
Figure 5.11 Morphological characters of Fusarium incarnatum M34 on C. calisaya. a,b. 7 days culture on PDA. b. Chlamydospore, d. Microconidia. Bar
a,b = 1 cm c,d = 10 μm
In order to resolve the identity of Fusarium sp. M34, strain M66 and M67, separate phylogenetic analysis based on partial EF1-
α sequence involving these three sequences with 42 sequences belonging to F. equisetti
–incarnatum was conducted. F. asiaticum strain NRRL 26156 GenBank accession number:
AF212452 was used as outgroup. The alignment of this dataset composed of 46
Figure 5.12 Morphological characters of Fusarium incarnatum M66 on C. calisaya. a,b. 7 days culture on PDA, c. Chlamydospore, d. Microconidia, e.
Phialides. Bar a,b = 1 cm c,d = 20 μm, e = 10 μm
Figure 5.13 Morphological characters of Fusarium incarnatum M67 on C. calisaya. a. 7 days culture on PDA, b. Chlamydospore. c. Conidiogenous cell, d.
Conidiophore, e. Phialid, f. Microconidia. Bar a = 1 cm b,c,d,e,f = 20 μm
sequences and 255 total characters included in the analysis, of which 144 charactersare constant, 43 characters are variable and parsimony-uninformative, 68
characters are parsimony-informative. All characters have equal weight. The best parsimonious tree was generated in 204 steps CI=0.667, RI=0.851, RC=0.567, HI
=0.333. The phylogenetic tree showed that Fusarium spp. M34, M66 and M67 formed monophyletic clade with F. incarnatum NRRL 34004 GQ505628 BS =
66.6 . This clade nested within the large monophyletic clade containing sequences belong to F. incarnatum sensu stricto BS=81.2 . Based on this
analysis, Fusarium spp. M34, M66 and M67 are determined as F. incarnatum. The phylogenetic study was supported by morphological observation on colony and
microscopic characteristics Fig 5.10
–5.15. The morphological difference between species were found Table 5.5.
Figure 5.14 Morphological characters of Fusarium solani M93 on C. calisaya. a,b. 7 days culture on PDA, b. Macroconidia and microconidia, c. Phialid. Bar
a = 1 cm c,d = 2 0 μm.
Figure 5.15 Morphological characters of Fusarium solani M97 on C. calisaya. a. 7 days culture on PDA. b. Chlamydospore, c. Phialid, d. Conidiophore,
e. Macroconidia and microconidia. Bar a = 1 cm b,c,d,e = 10 μm
Table 5.5 Morphological comparison of the structures of three endophytic fungi Fusarium species associated with C. calisaya
Morphological F. incarnatum M66
F. incarnatum M34 F. incarnatum M67
F. oxysporum M16 F. solani M93
F. solani M97 characteristics
Microconidia 8.7−11.04 × 13.69−45.23
2.93−3.88 × 6.12−8.63 2.47−4.65×6.04−21.56
8.75−12.89 × 25.41− 34.63 2.90 − 5.21 x 13.18−17.21
2.90−8.72 X 8.89−20.90 oblong, aseptate
oval, aseptat oblong, aseptat
ovale, aseptate ovale, aseptat
ovale, aseptat Macroconidia
− 3.61−4.15× 11.9−20.9
− 8.49−11.75× 31.92−70.43
19.91 –38.63×3.35–5.91
3.35−11.36×19.91−62.64 −
oblong-elliptical, aseptat −
obovate, 3 –6 septat
obovate, septat 4-6 obovate, aseptat
Conidiophore −
− long and single
− long and single
long and single Phialides µm
Monophialide −
Monophialide −
monophialide monophialide
1.4 ×4.3 −
1.4× 6.4 −
3.3 ×5.86 2.7×9.31
Chlamydospore present, globular,
present, obovoid present, globular,
present, globular, present, ovale,
present chain+ or single, intercalar and terimal
Intercalar Intercalar
intercalar, terminal intercalar
ovale, intercalar 5.3−7.9 ×7.1−9.7
6.3×8.5 3.1−4.3× 3.3−4.9
6.1−10.6 ×5.4−11.4 4.5−6.5 ×7.3−8.1
4.6−8.8×6.5−9.2 Colour
white to pink white to pink
White purpledark purple
white to cream white
Size cm of colony 4.8
4.2 5.2
4.3 5.8
4.8 Mycelium
cottony, aerial cottony, aerial
cottony, aerial cottony, immersed
cottony, aerial cottony, immersed
57
DISCUSSION
Cercospora
The first published paper on Cer. cinchonae of the quina plant was written by Ellis Everth 1887, then Crous Braun 2003 has changed its name to
Pseudocercospora cinchonae. Boedijn 1962 had described two new species Cer. cinchonicoea and Cer. cinchonicola based on specimen from Quina plant in Indonesia.
Braun 2001 moved Cer. cinchonicola to Pseudocercospora cinchonicola. All this species areas plant pathogens. In this study, Cercospora sp. M18A and M18B that were
isolated from healthy C. calisaya petiole in Java, Indonesia are endophytes. Based on phylogenetic analysis with the ITS data matrix contained 10 genera of Cercosporoid s.
str, Cercospora sp. M18A and 18B form monophyletic within the genus of Cercospora. In fact, Cercospora sp. M18A and M18B to which the name Cercospora
sp. applies, apparently are not Cercospora cinchonae Ellis Everth 1887 and Cer. cinchonicola, Boedijn 1962, since the last two names have characteristics of
Pseudocercospora. Therefore Cercospora M18A, M18B were considered as a new species, Cer. cinchonae. These species was the first reported endophytic Cercospora
in C. calisaya from Indonesia.
The combination of the morphological and phylogenetic elucidation of new proposed taxa in Cercopsora is quite important to misidentification. The phylogenetic
tree generated from maximum parsimony analysis by combining five genes loci rDNA region showed that the monophyletic of Cercospora with 55 bootstrap value. The
Cercospora sp M18A and Cercospora sp. M18B formed a monophyletic clade with 100 bootsrap value. These strains were included in one species. This clade appeared
as a sister group C. cf. malloti, C. kikuchii, C. cf. richardiicola, C. cf. sigesbeckiae clade with 65 bootstrap value which indicates a close relatioship between one and
others.
The phylogenetic tree sequences showed that two sequences of Cercospora sp. 18A and 18B from petiole formed independent clade which separated from other
Cercospora sequences included in the analysis 65 BS. The phylogenetic tree is obtained by using the combined sequence data of five genomic loci. The Cercospora
sp. M18A and M18B are distinct genetically from other Cercospora sequences. Therefore, members of this clade are proposed as a new species based on phylogenetic
analysis.
Taylor et al. 2000 developed the Genealogical Concordance Phylogenetic Species Recognition GCPSR concept to definethe limits of sexual species, using the
phylogenetic concordance of multiple unlinked genes. This concept has proved greatly useful in fungi, because it is more finely discriminating than other species concepts, as
several species can not be recognised due to the lack of distinguishing morphological characters or mycelia sterilia Cai et al. 2011.
Cercospora sp. M18A and M18B genes had been analysed with ITS, EF, CAL, ACT and HIS because it was not identity with
morphological characters. The adoption of genealogical concordance for species
recognition in Cercospora sp. to distinguish species otherwise possible to identify due to mycelia sterilia.
Cercospora cinchonicola caused disease in the Quina plant was first reported in Indonesia and identification based on morphological characters
by Boedijn 1962.
Morphological characteristics of Cer. cinchonicola were stromata small, flattened, blackish brown 15−40 µm in diameter, fascicles fairly dense. Conidiophore brown,
with a short side branch, sparingly septate, geniculate, tip bluntly rounded 20−60 x 3−3.5 µm. Conidia pale olivaceous, narrowly obclave, mildy curved, indistincly
multiseptate, base truncate, tip rounded, 64−110 × 3−4 µm. Cercospora sp. M18 having Stromata small, flattened, brown. Conidiophores clustered, arising from the
upper cells of stromata, straight, subcylindrical to flexuous, unbranches, 40−100 × 5−7
µm, septate, thin-walled, smooth, brown; conidia was not observated. Unfortunately, in this study conidia was not found in Cercospora sp. as morphological characteristics.
Therefore, Cercospora can not be identified as the same species with Cer. cinchonicola, because they were found within the same plant host.
Many Cercospora species that are morphologically similar proved to be genetically distinct, and several
isolates that were formerly identified based on their host, were show to represent different taxa To-Anun et al. 2011.
Boedijn 1962 pointed out that the large number of the Cercospora species was due to the presence of a wide variety of vascular plants in Indonesia. He reported
90 species of Cercospora from 109 host plant species, including those from Cinchona plant. In Thailand, various hosts such as crops, weeds and ornamentals plants could
associate with cercosporoid fungi. There were one hundred and six species have been recorded from several locations Meeboon et al. 2007; Nakashima et al. 2007.
Cercospora species are frequently classified according to host. Highly host-specificity species, e.g. Cercospora beticola from sugar beet or Beta vulgaris Den Breeyen et al.
2006 , Cercospora christellae from weed Christella parasitica from northern Thailand To-anun et al. 2010. Cercopsora habenariicola from leaf of Habenaria susannae L.
R. Br. Orchidaceae Meeboon et al. 2007 and Cercospora brassicicola from leaves of Brassica oleracea L. Brassicaceae in Thailand Meeboon et al. 2005 were noted.
Groenewald et al. 2005 considered that Cercopsora was not host spesific because some of Cercospora can infect a wide host range e.g. C. apii complex can infect Apium
graveolens celery and Beta vulgaris sugar beet, Cercospora cf. citrulina can infect Musa sp., Citrullus lanatus, and Momordica charanthia. Groenewald et al. 2012
stated that some species were found to be limited to a specific host genus and other genus have a wide host range.
The stimulate sporulation media had given banana leaves and plant organs quinine on the surface of the agar media could not form sporulation. More detailed
studies are required to describe the morphological characters of Cercospora sp. M18 as endophytic fungi. In this study Cercopora sp. could not be found conidia in the
artifisial media. Several problematic endophytic fungi could not distinct morphological characters or sterility, e.g., Diaporthe Gomes et al. 2013, Colletotrichum Damm et
al. 2012. Vathakos and Walters 1979 observed that Cercospora kikuchii in the Senescent Soybean Plant Agar SSPA could be exposed abudant sporulation on
alternating dark and light periods and 8 d to Gro-lux lamp, but it did not occur in continuous darkness. More conidia can be produced if transfer of spores rather than
mycelium resulted in cultures. Goode Brown 1979 postulated that the ability of some Cercospora spp. to sporulate for a few generations in artificial culture indicated
that those isolates have a genetic component for sporulation.
The Cercospora are considered as plant pathogens, C. kikuchii is the only species known both as plant pathogen and endophyte Tales 2011. Little information
of endophytic Cercospora were found. During study of endophytic fungal diversity associated with C. calisaya, the most important medicinal plant e.g. antimalaria,
antibacterial, etc Maehara et al. 2001, Skogman et al. 2012, Wolf et al. 2002, two isolates of Cercospora-like were found. The existence of some endophytic fungi in the
stem and bark of quinine plant have been reported by several researchers in Indonesia, however, none of those researchers reported the endophytic Cercospora. Therefore, the
findings of endophytic Cercospora from C. calisaya was interesting and to analyze its potential for alkaloid production, particularly quinine, quinidine, conchonine, and
cinchonidine.
Diaporthe
The identification of genera Diaporthe using ITS sequence is problematics. The application of molecular phylogenetic analysis has resolved problems in determination
of DiaporthePhomopsis species complex Rensburg et al. 2006; Udayanga et al. 2012; Gomes et al. 2013. Majority of these reports involving utilization of sequences from
18S, ITS and 28S rDNA region Zhang et al. 1998; Niekerk et al. 2005, and in combination with DNA sequence generated from part of the elongation factor 1-
α gene EF1-
α Rensburg et al. 2006; Thompson et al. 2011, β–tubulin and calmodulin Gomes et al. 2013. Gomes et al. 2013 reported that several taxa of Diaporthe are
host-specific, while some species have wide host ranges. These include members of Diaporthe living in plant tissue as endophyte. Therefore, identification of dominant
endophytic Diaporthe spp. is of importance.
Identification of the Diaporthe into species require additional genes other than ITS.
Identification based on ITS rDNA is commonly used to identify to the species level, especially if the endophytic fungus does not sporulate Zhang et al. 1998.
Identification by ITS sequences can be used as a morphological identification verification, against unidentified endophytic fungus based on morphological characters
Huang et al. 2009.
Therefore, further identification of Diaporthe spp. is done by using a combination of rDNA ITS regions and EF1-
gene. Phylogenetic analysis showed that only 17 of the 39 strains of endophyte Diaporthe found can be named species,
while the remaining 22 strains can not be named species. Multigene analysis led to the name of the species based on the ITS approach can not be used. Gomes et al. 2013
use more than one gene for identification of molecular Diaporthe using a combination of ITS sequences, EF1-
, ACT Actin, CAL Calmodulin and HIS Histon. The results of molecular analyzes of strains Diaporthe spp. by using
combination of ITS and EF1- α left 22 strains unidentified. Identification is based on a
combination approach ITS and EF can change the status of naming species based on ITS approach. The name of D. litichola M78, D. pseudomangiferae M24, D.
phaseolorum M10 and M40, D. ganjae M71 unchanged, whereas D. helianthi M21
becomes D. litchicola M21, D. psoraleae-pinnatae M32, M84, and M94 under ITS phylogenetic tree are moved into D. rhoina M32, M84, and M94. Further, Phomopsis
palmicola M11 moved into D. gardeniae M11, D. beckhausii became D. gardeniae M35 M35, D. beckhausii M54 became D. cynaroidis M54, while Diaporthe sp. M12,
M15, M22 are identified as D. gardeniae M12, M15 and M22, Diaporthe sp. M90 become D. endophytica M90. Diaporthe sp. M89 is identified as D. rhoina M89.
Identification using multigene approach is usually more accurate than identification by a single gene.
The use of molecular identification through polyphasic approach by using multigene and secondary metabolites analysis is supposed to enhance the accuracy of
Diaporthe spp. identification. Meanwhile, profile clustering is not in accordance with phylogenetic groupings. This indicates that alkaloids profile can not be considered as
criterion included in polyphasic approach as the alkaloid production is each strain- dependent.
No new species can be proposed for any Diaporthe spp. in the phylogenetic study.
The current study on the six Diaporthe strains included for cinchona alkaloids analyses revealed that at least, three distinct lineages of Diaporthe living inside the
tissue of C. calisaya, i.e., D. cinchonae inhabiting branch and petiole, D. endophytica inhabiting leaf, and Diaporthe sp. InaCC-F235 inhabiting fruit. Diaporthe
endophytica was also reported as common endophyte inhabiting S. terebinthifolia leaf, M. ilicifolia petiole, and G. max seed in Brazil Gomes et al. 2013. The
existence of more than one species of endophytic Diaporthe in the single host plant species was not uncommon as several authors previously had reported. For example,
about 15 Phomopsis spp. were determined from grapevines Niekerk et al. 2005, at least four Diaporthe taxa occurred in fennel Foeniculum vulgare Santos Phillips
2009, as well as in soybean Glycine max Santos et al. 2011 and in sunflower Helianthus annuus Thompson et al. 2011, respectively. These information
demonstrate that implementation of host-based species concept in determination DiaporthePhomopsis must be avoided Hyde et al. 2010. The existence of more than
one species of DiaporthePhomopsis within one host or the possibility of single species of DiaporthePhomopsis has more than one host will definitely affects the management
of plant diseases caused by this fungus in the future.
Although the phylogenetic analysis clearly showed differences among D. cinchonae, D. endophytica, and Diaporthe sp. strain InaCC-F235, however, the
morphological characteristics of these isolates are similar. Slightly differences were found on pycnidial size and β-conidia. The size of β-conidia of D. cinchonae 23.6–
27.1 × 1.6 –2.1 µm was found slightly larger than D. endophytica 17.4–20.7 × 1.9–
2.4 µm and Diaporthe sp. strain InaCC-F235 22.1 –24.9 × 1.3–1.6 µm. However, the
colonies characteristics of these 3 species were apparently distinct. Diaporthe cinchonae formed wavy and zonate concentric radial textures, which was distinct from
isolates of D. endophytica and Diaporthe sp. strain InaCC-F235 having concentric radial texture with non-wavy edge and concentric with variegated or irregular wavy at
the edge, respectively. These data showed that morphological characters such as conidiomatal structure and conidial size are unreliable in determining
DiaporthePhomopsis complex into species level. In addition, many species of DiaporthePhomopsis
only produce α- or β-conidia, and only about 20 of Phomopsis species have a known teleomorph Rehner Uecker 1994.
The molecular phylogenetic analysis is greatly needed to advance more specific genetic evidence to support taxonomic differences, because of limited morphological
characters in differentiating species. Gomes et al. 2013 also previously reported that many morphologically similar DiaporthePhomopsis species were proved to be
genetically distinct. The combination of morphology and cultural characters with molecular analyses will raise precise identification and description for
DiaporthePhomopsis species from a range of host plants. The current study also indicates that tissue types seems to be important microhabitats for specific
DiaporthePhomopsis inhabiting the same host. However, more sequences are necessary to be included in the analysis to justify this hypothesis.
Due to the considerable overlap of morphological features among the available species within DiaporthePhomopsis, Santos and Phillips 2009 suggested that the
phylogenetic species concept developed by Taylor et al. 2000 is probably best fits to determine members of this fungal group into species level. Several problematic
endophytic fungi lacking of distinct morphological characters or sterility, e.g., Diaporthe Gomes et al. 2013, Colletotrichum Damm et al. 2012, and Phyllosticta
Glienke et al. 2011 have been resolved. However, controversies regarding how many genes sufficient for determination of fungi into species level using this approach are
still continue. In DiaporthePhomopsis complex, phylogenetic analyses of sequence from ITS region as primary region, and in combination with HIS or TUB regions were
recommended Udayanga et al. 2012; Gomes et al. 2013. Combination of ITS and TEF1 sequences were also common in identification of DiaporthePhomopsis complex
Ash et al. 2010; Santos et al. 2011; Thompson et al. 2011. In the case of Diaporthe sp. strain InaCC-F235, identification using combined sequences of
ITS and TEF1‒ was proven insufficient. Although the phylogenetic tree sequences showed that
Diaporthe sp. strain InaCCF235 did not link to any other sequences included in the analysis, however, it is not enough evidence to propose this strain as a novel species.
Furthermore, slight morphological differences of this strain to closely related species have also been found insufficient to support phylogenetic data for species
determination. Additional sequence data from morphologically similar endophytic Diaporthe isolates from C. calisaya or additional sequence data from other gene
regions are necessary to resolve the identity of this endophytic fungus. The phylogenetic tree generated from combination of ITS and part of EF1-
α sequence showed that four sequences of D. cinchonae form an independent clade within the large
clade containing Diaporthe sp. strain InaCC-F235, D. hongkongensis, D. arecae, D. pseudophoenicicola, D. pseudomangiferae, D. eugeniae, D. arengae, D. musigena, D.
perseae, D. arecae, and several species of Diaporthe on different hosts. Within this clade, D. hongkongensis was the closest sequence to D. cinchonae. Based on
morphological characteristics, D. cinchonae differs from D. hongkongensis by having larger
pycnidia vs. up to 200 μm diam. of D. hongkongensis, longer beta conidia vs.
18 –22 × 1.5–2 μm of D. hongkongensis, and lacking of gamma conidia at least during
this study. The phylogenetic tree generated from combination of ITS and part of EF 1-
α sequence analyses clearly showed that this strain belonging to D. endophytica 81
BS. The type species of D. endophytica was first published from the leaf of Schinus terebinthifolia Raddi
Anacardiaceae Gomes et al. 2013 . This species is also
recorded as endophyte in petiole of Maytenus ilicifolia Mart. ex Reissek Celastraceae
and in seed of Glycine max L. Merr. Fabaceae. This is the first report of D. endophytica in C. calisaya from Indonesia.
Fusarium
Phylogenetic analysis of quinine-producing Fusarium species showed that there are more than one species of the genus Fusarium exist as endophyte within plant
tissues of C. calisaya. The finding of more than one species from a single fungal genus occupy the same species of host plant, in fact, was not uncommon as several authors
had previously reported Niekerk et al. 2005; Santos Phillips 2009; Santos et al. 2011; Thompson et al. 2011. The current study also found that a single species of
endophyte can occupies different type of plant organs as showed by several morphotypes of F. incarnatum isolated from petiole strain M34, fruit strain M66
and bark strain M67.
Fusarium is hyphomycetes soil-borne fungi that causes the most economically important plant diseases. Fusarium sp. is cosmopolitan, occurs on a wide range of host
plants as plant pathogenic, such as tomatoes Kim et al 2007; Ignjatov et al. 2012, banana Dita et al. 2010, maize Rodriguez et al. 2007. Fusarium also causes diseases
in corn, figs, pine, rice and sorgum Nelson et al. 1993. Some species are known to cause diseases in animals and humans, which is important to study because those
species remove mycotoxins Monds et al. 2005.
The identification of the occurrences of endophytic Fusarium sp. in C. calisaya has not gained much attention, compared to pathogenic Fusarium sp., although there
have been reports on Fusarium sp. as the symptom of infections in several types of plant. Therefore, this study was conducted to isolate endophytic Fusarium sp. from all
parts of the plant organ of C. calisaya. The association between the endophytic fungus and the plant host may also switch from mutualistic to pathogenic depending on several
factors Moricca Ragazzi 2008. The endophyte fungi can infect healthy tissues as biotrophs, but fungus may infect the host after a latent period or become a saprophyte
when then host plant dies. Therefore, the association between the fungal endophyte and the host plant is like transitory Bacon Yates 2006.
The identification of the occurences of Fusarium spp. in different forms is often confusing due to their similar morphological features. Morphological characters to
identify the species is problematic because mycelia pigmentation, shape and size of conidia are unstable and dependent on the composition of media and environmental
condition Guo et al. 2001. The differentiation of genes in DNA sequences has been used to support morphological identification of Fusarium sp. Phylogenetic analysis of
DNA sequences has been used to distinguish genetic relationships among closely
related Fusarium sp. Molecular techniques for fungal identification within species have been used via the ITS region and other gene sequences.
O’Donnell et al. 1998, Watanabe et al. 2011 stated that the rDNA clu
ster region β-tubulin gene β-tub, the elongation factor 1-
α gene EF1-α, and aminoadipate reductase gene lys2, have been used as genetic markers for the phylogenetic analysis of Fusarium sp.
The most of Fusarium are classified as saprotrophic, phytopathogenic, and endophyte Barik et al. 2010. Several Fusarium spp. isolated as endophyte from
medicinal plants and other plants have been reported to produce metabolites activity, such as grass family Gramineae Sieber 2002, Camptotheca acuminate Wang et al.
2006, Pinus massoniana Lamb Wang et al. 2008, Dracaena cambodiana and Aquilaria sinensis Gong Guo 200, Annona squamosa, Taxus chinensis Deng et al.
2009, and Axonopus compressus Zakaria Ning 2013. Endophytic F. oxysporum and F. solani have been found on the roots of several vegetables Kim et al. 2007,
while F. solani have been found on the roots of Rhizophoraceae mangrove trees Xing Guo 2011. There has been no report of the research on endophytic fungus Fusarium
sp. and its potential to produce quinine, which was isolated from the entire plant organs of C. calisaya.
CONCLUSION
The identity reconfirmation Cercospora based on amplyfy region ITS, EF1- α
ACT, CAL and HIS analysis, Cercospora was identifified as Cercospora sp. The genera of Diaporthe and Fusarium based on ITS and EF1-
α analysis were identified Diaporthe sp. D. cinchonae, D. endophytica, F. incarnatum, F. oxysporum, and F.
solani. The Cercospora sp., Diaporthe sp. and D. cinchonae are candidate for new species, but appropriate morphological features are needed to support this proposal.
Alkaloid profile clustering of Diaporthe spp. is not in accordance with phylogenetic groupings.
6 GENERAL DISCUSSION BIODIVERSITY OF ENDOPHYTIC FUNGI FROM