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Penicillium coffeae, a new endophytic species isolated from a coffee plant and its phylogenetic relationship to P. fellutanum, P. thiersii and P. brocae based on parsimony analysis of multilocus DNA sequences

     Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604

Fernando E. Vega
Francisco Posada

     Insect Biocontrol Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705

Chifumi Nagai

     Hawaii Agriculture Research Center (HARC), 99-193 Aiea Heights Drive, Suite 300 Aiea, Hawaii 96701-3911

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

Penicillium coffeae is described as a novel endophyte isolated from a Coffea arabica L. plant in Hawaii. The species is slow growing with short, vesiculate, monoverticillate conidiophores. Phylogenetic analysis using three loci shows that P. coffeae forms a strongly supported clade with P. fellutanum, P. charlesii, P. chermesinum, P. indicum, P. phoeniceum and P. brocae. Phenotypic ally these species are quite similar but can be distinguished. The EF-1 gene from P. fellutanum, P. charlesii, P. chermesinum and P. indicum lack introns, P. coffeae and P. phoeniceum have a previously unknown intron at codon 20 and P. brocae and P. thiersii isolates have a single intron at codon 26. The most parsimonious interpretation of intron changes on the strongly supported phylogenetic tree requires the gain of a novel intron at position 20 and loss of intron 26 to arrive at the current distribution of introns in this gene. This is one of only a few examples of intron gain in genes.

Key words: Penicillium phoeniceum, Penicillium indicum, Penicillium ebenbitarianum, Penicillium atrovirens var nigrocastaneum, molecular systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Although coffee has been commercially planted for hundreds of years and the amount of published research on coffee production is staggering, very little is known about microorganisms present within the plant. Of paramount importance among such organisms are fungal and bacterial endophytes that live in the intercellular spaces of plants but cause no symptoms of disease. Fungal endophytes are known to produce bioactive products (Strobel 2003) and have been shown in some cases to have positive effects on the plant (Clay 1994, Bacon and White 2000, Azevedo et al 2000, Arnold et al 2003).

We have been studying the fungal endophyte diversity in coffee, and among the hundreds of fungi we have isolated there are several Penicillium species that we will report elsewhere (Vega et al in preparation). This finding might have practical importance in coffee production due to the wide array of metabolites produced by Penicillium species (Cole and Schweikert 2003, Cole et al 2003). Various Penicillium species have been reported as endophytes in plants (Spurr and Welty 1975, Collado et al 1999, Shaukat and Siddiqui 2001, Larran et al 2001, Cao et al 2002, Maria and Sridhar 2003, Yong et al 2003, dos Santos et al 2003) and in coffee seeds (Batista et al 2003, Reynaud et al 2003).

Four of the Penicillium isolates obtained in this study were highly similar to each other but were not assignable to any described species (Raper and Thom 1949, Pitt 1980, Ramirez 1982). BLASTN (Altschul et al 1997) searches of GenBank, using the internal transcribed spacer region (ITS) and large subunit (lsu) ribosomal DNA (rDNA) sequences from these four isolates, failed to reveal any closely related species. Accordingly, we computed phylogenetic trees from ITS and lsu rDNA sequences to place these isolates in the general Penicillium tree (Peterson 2000). These isolates are most closely related to P. charlesii, P. fellutanum, P. phoeniceum, P. indicum, P. brocae and the recently described species P. thiersii. Because they are phylogenetically distinct from known species, we describe these isolates as the new species Penicillium coffeae.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
The fungal cultures used are provided (TABLE I) and are available upon request from the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois.

Phenotypic analysis.— – Cultures were grown on Czapek yeast-extract agar (CYA), malt-extract agar (MEA) and glycerol-nitrate agar (G25N) under the conditions recommended by Pitt (1980). Colonies were observed with a dissecting microscope and a compound microscope (Zeiss axioskope) equipped with phase and differential interference contrast (DIC). Scanning electron microscopy was performed on samples fixed with osmium tetroxide, dehydrated with acetone, critical point dried and coated with gold-palladium (Peterson 1992). Colony color names are based on the Ridgway (1912) nomenclature. Microscopic measurements were analyzed statistically with Excel (Microsoft, Bellevue, Washington). Photographs were taken with a Kodak 420B digital camera with macrolenses and with an adaptor tube for the axioskope.

DNA isolation, amplification and sequencing.— – Cultures were grown either on agar slants in tubes or on 4.5 cm diam Petri dishes containing MEA. After 7–10 d mycelium was scraped from the colonies, placed in a disposable tube with acid-washed glass beads and buffer, followed by vortex mixing (60 s) to break the cell walls. Proteins were extracted with phenol : chloroform (1:1); the aqueous phase was isolated by centrifugation for 5 min at 2000 g, and nucleic acid were precipitated by addition of 1.3 volume 95% ethanol. Nucleic acids were dissolved in TE buffer and adsorbed to silica particles in the presence of concentrated NaI (Gene-Clean, Qbiogene Inc., Carlsbad, California) and eluted in TE buffer. DNA solutions were stored at –20 C.

The ITS and partial large subunit rDNA (ID region) was amplified with primers ITS-5 (White et al 1990) and D2R in the protocol of Peterson et al (2004). The calmodulin gene (CAL) was amplified with primer CF1d and CF4, and translation elongation factor 1- (EF 1-) was amplified with primers EF1b and EF6 in the procedures of Peterson et al (2004).

Amplified gene fragments were purified with the Millipore Multiscreen PCR system as detailed by the manufacturer (Millipore, Billerica, Massachusetts). Purified fragments were sequenced with the terminal primers used in amplification plus internal primers (Peterson et al 2004) and fluorescent dye labeled dideoxy nucleotide terminators in the Applied Biosystems Dye-deoxy sequencing kits. Sequences were read on an Applied Biosystems model 377, 3100 or 3730 DNA sequencer (Applied Biosystems Inc., Foster City, California). Sequencing procedures were performed in accordance with the manufacturer’s instructions.

Phylogenetic analysis.— – DNA sequences were aligned with Clustal W (Thompson et al 1994) followed by visual corrections with a text editor. Modeltest 3.06 (Posada and Crandall 1998) was used to determine the evolutionary model that best fit the data. Trees were calculated with PAUP* 4.0 ß10 (Swofford 2003) with maximum parsimony or maximum likelihood criterion, random addition order and TBR branch swapping. Bootstrap values were calculated using heuristic search and 1000 samples. The congruence of alternative trees was determined with the Kishino-Hasagawa test in PAUP*. Trees were viewed with TreeView (Page 1996) and formatted for publication with CorelDraw 9.

	RESULTS

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Several species of Penicillium and other genera were isolated as endophytes of coffee. The details of those isolates will be published elsewhere (Vega et al in preparation). The new species was isolated from only one peduncle in coffee plant K033 at the Kunia Field Station. Four separate isolates were obtained, but it seems likely that these isolates are clonally related. The DNA sequences from the four isolates were identical, further suggesting that they are clonal.

The ID region sequences range was 1129–1152 nt in length. The aligned dataset of 1156 nt included 1008 constant and 103 parsimony informative characters. Heuristic search of the dataset produced 10 equally parsimonious trees of 159 steps with CI = 0.8113 and RC = 0.7780. The best model for this data was GTR + I + , with I = 0.7023 and the shape parameter alpha = 0.6682. The sequences are deposited in GenBank with accession numbers AF033399 [GenBank] , AF125936 [GenBank] , AF484391 [GenBank] –AF484399 [GenBank] and AY742692 [GenBank] –AY742708 [GenBank] .

Calmodulin sequence lengths were 677–704 nt with an aligned length of 737 nt. All length differences in CAL were due to indels in the introns. All isolates possessed intron sequences at codon 20 (phase 0), and at codons 26, 68 and 139 (all phase 1). Introns at phase 0 are inserted between codons, phase 1 between bases 1 and 2 of a codon and phase 2 between bases 2 and 3. The amino acid sequences predicted from the coding regions of species in this study were identical although those DNA sequences differed. Parsimony analysis of the coding region was performed with the conditions specified above. The data set included 307 constant and 66 parsimony informative positions and gave two equally parsimonious trees of 113 steps with CI = 0.7434 and RC = 0.7030. The best model for this data was GTR + I + with I = 0.6749 and the shape parameter = 0.1643. The sequences are deposited in GenBank with accession numbers AY741726 [GenBank] –AY741754 [GenBank] .

Elongation factor-1 sequences were aligned in a data set of length 742 nt, with the individual sequences ranging from 583–684 nt. Isolates of P. coffeae and P. phoeniceum each possessed an intron inserted at codon 20 (phase 1), P. thiersii and P. brocae isolates contained a single intron at codon 26 (phase 1) and P. charlesii, P. indicum, P. chermesinum and P. fellutanum isolates contained no intron sequences in the region sequenced. The intron at codon 20 has not been reported previously. Amino acid sequences predicted from the coding region DNA reveal EF-1 proteins with amino acid differences at amino acid positions 77, 79, 155, 157, 179, 196 and 198. Most amino acid differences are between the outgroup species and the ingroup. Introns were excluded from analysis and the data set included 598 constant and 57 parsimony informative characters. Heuristic search of the data produced two equally parsimonious trees of 97 steps with CI = 0.6598 and RC = 0.6110. The best model for these data was the GTR + I + with I = 0.7122 and the shape parameter = 0.4780. The sequences are deposited in GenBank with accession numbers AY741755 [GenBank] –AY741783 [GenBank] .

Reduced datasets containing only the ex type isolates were constructed and evaluated with PAUP*. ID data produced three most parsimonious trees; CAL data produced a single most parsimonious tree; and EF-1 data produced a single most parsimonious tree. Noncongruence of the trees was statistically insignificant as assessed with the Kishino-Hasegawa test and the GTR + I + model. The most parsimonious trees from EF-1 and CAL are presented (FIG. 1), with predicted losses and gains of introns that would explain the current distribution of introns among the species. Tree CAL is the most parsimonious requiring a single loss of intron 26 and a single gain of intron 20. Because the incongruence was statistically insignificant, the data were concatenated into a single dataset. Its analysis produced the single tree (FIG. 2) that has the same configuration as tree CAL (FIG. 1).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Single most parsimonious trees obtained from the calmodulin (CAL, left) and elongation factor-1 (EF-1, right) datasets. The trees are noncongruent, but statistical analysis does not favor one topology over the other. Hypothesized gains and losses of introns that would explain the current distribution of EF-1 introns are labeled on the trees. The calmodulin based tree is favored because it requires the smallest number of intron gains and losses. TreeBase No. SN2019.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Single most parsimonious tree based on concatenated data from the ITS and partial large subunit rDNA (ID region), calmodulin gene (CAL) and elongation factor-1 loci (EF1-). Bootstrap sample percentages supporting branches are placed on internodes. Penicillium atrovirens and P. fellutanum var nigrocastaneum are synonyms of P. charlesii, and P. ebenbitarianum is a synonym of P. fellutanum. Penicillium coffeae is most closely related to Penicillium phoeniceum. Ex type or ex neotype cultures are denoted by a superscript T or NT. TreeBase No. SN2019.

	TAXONOMY

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View larger version (53K): [in this window] [in a new window]   FIGS. 3–8. Penicillium coffeae NRRL 35363. 3. Colonies grown 7 d on CYA (left) and MEA (right). 4. Close-up view of a CYA grown colony showing sulcation and scalloped margin. 5. Close-up view of an MEA-grown colony showing the small aerial portion of the colony and the larger submerged portion of the colony. 6. SEM of a mature conidiophore showing the smooth stalk and crowded whorl of phialides. 7. Light micrograph (DIC) showing the inflated vesicle typical of conidiophores in this species. 8. SEM showing faintly roughened spherical conidia. Bar in FIGS. 6, 7 = 10 µm; FIG. 8 = 5 µm.

Colonies after 7 d on CYA 11–17 mm diam (FIGS. 3, 4), radially sulcate, lanose, lacking exudate, color variable with state of sporulation from grayish-olive to Artemisia or Gnaphalium green, margin scalloped, reverse is light drab to light grayish, soluble pigments and sclerotia are absent. Colonies on MEA after 7 d 11–13 mm diam (FIGS. 3, 5), plane, velutinous, lacking exudate, celandine green, with heavy sporulation, submerged growth accounting for two-thirds of colony diameter, reverse colonial buff to ivory yellow, soluble pigments and sclerotia are absent. Conidiophores (FIGS. 6, 7) similar on both media arising from basal hyphae, monoverticillate, smooth 50–200 x 2.0–2.5 µm with a terminal vesicle 3–8 µm diam, conidiogenous cells ampuliform 8.5–12 x 2.0–2.5 µm in whorls of 6–10 or more producing smooth spherical conidia 2.5–3.5 µm diam (FIG. 8).

Etymology. Epithet is based on the host plant.

HOLOTYPE: BPI863480 here designated, consists of colonies of NRRL 35363 grown 7 d on CYA and MEA agars, affixed to a slide mailer and dried.

Penicillium coffeae is most readily recognized by the monoverticillate, vesiculate conidiophores, small colony diameter and the large proportion of submerged colony growth on MEA. In this last character it differs from the other species considered here, and this appears to be a good character for distinguishing the species.

Cultures of P. charlesii and P. fellutanum were examined with the diagnostic media of Pitt (1980). Most characters recorded (TABLE II) were not sufficiently distinct to differentiate between these two species, a situation that led Pitt (1980) to place the names in synonymy. However we found that conidia of P. charlesii were elliptical, averaging 3.1 x 2.7 µm, and finely roughened while the conidia of P. fellutanum isolates were nearly spherical, 2.7 x 2.5, µm and smooth. Penicillium indicum could be distinguished on the basis of greater colony diameter on CYA and MEA at 25 C and heavy clear exudate on CYA (TABLE II). Penicillium chermesinum made relatively large diameter colonies on CYA and MEA but produced slightly smaller, more elliptical conidia than P. indicum. Penicillium phoeniceum produced small colonies, as did P. fellutanum, P. charlesii and P. coffeae but could be distinguished from P. charlesii and P. fellutanum by being strictly monoverticillate, producing slightly larger phialides and spherical 3.1–3.2 µm diam conidia. Penicillium coffeae produced colonies composed mostly of submerged growth on MEA compared to the other species whose submerged and surface growths were of nearly equal diameter. CYA colonies tended to be dull grayish blue and produced smooth, spherical conidia of 2.6 µm diam.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

Penicillium fellutanum and P. charlesii are separated on the basis of subtle phenotypic characters, but more observations using different media and growth conditions might reveal additional and more distinctive characters. Pitt et al (2000) treated P. charlesii as a synonym of P. fellutanum. These sibling species are isolated genetically and have diagnosable characters, and we recognize them as species. Ramirez (1982) considered P. fellutanum to be strictly monoverticillate, while P. charlesii contained furcate and monoverticillate conidiophores. We have observed both monoverticillate and furcate conidiophores in isolates of each species and have been unable to separate the species on the basis of conidiophore complexity. The isolate of P. atrovirens and the isolate of P. fellutanum var. nigrocastaneum are indistinguishable from P. charlesii, and they are synonyms of P. charlesii. The ex type isolate of P. ebenbitarianum is on the same terminal branch as P. fellutanum and is a synonym of that species. These synonymies are in agreement with Pitt et al (2000) except that we accept P. charlesii as a distinct species.

The phylogenetic placements of P. indicum and P. phoeniceum had not been established previously with DNA sequence data, but our data show (FIG. 2) that they form a strongly supported clade along with P. coffeae, P. fellutanum, P. chermesinum and P. charlesii. The overall phenotypic similarity of these species is striking (TABLE II). Pitt et al (2000) consider P. indicum to be a synonym of P. chermesinum. These two species are siblings, but the three genetic loci used here show that they are distinct species. There are phenotypic distinctions (TABLE II) that can be used to distinguish them. Phenotypic similarity among closely related species in well defined lineages was observed in the P. miczynskii clade (Peterson et al 2004) and the P. brevicompactum clade (Peterson 2004).

Pitt et al (2000) list P. phoeniceum as the anamorphic state of Eupenicillium cinnamopurpureum. A comparison of ID region DNA sequences from the type isolates of each species reveals a three base-length difference and a 94% similarity in Clustal W alignment. In an alignment of the EF-1 genes, E. cinnamopurpureum (NRRL 3326, ex type culture) has introns at codon 26 (phase 1) and codon 45 (phase 0) versus the total lack of introns in the P. phoeniceum gene and the overall similarity of the coding region sequences is 70%. The phenotypic similarity of P. phoeniceum and the anamorphic state of E. cinnamopurpureum result from convergent evolution, and these two species are distinct. Peterson and Sigler (2002) found a similar situation for P. pullum (syn. = P. fuscum), which on phenotypic grounds once was considered to be the anamorph of Eupenicillium pinetorum (Stolk and Samson 1983), but molecular genetic studies showed it to be distinct. Convergent evolution has made it nearly impossible to assign anamorph-teleomorph connections purely on the basis of phenotype.

There is incongruence of the trees generated with CAL and EF-1 datasets. However, when the datasets were tested against the two tree topologies, neither topology was significantly better than the other for either dataset. The stochastic nature of nucleotide substitution makes it likely that the rate of change will be nonlinear over relatively short time spans and we believe this is the cause of the incongruence. This hypothesis could be tested by sequencing additional genes that have a higher intrinsic rate of change, and that could hypothetically provide linearity over a smaller time span.

Novel intron gains in protein coding DNA was a controversial topic until ver y recently (Logsdon 2004), and there is little certainty about the mechanisms of intron loss and gain. For this reason we have chosen the most parsimonious explanation for the present day distribution of the introns in the EF-1 gene (FIG. 1) as also was done in the P. miczynskii clade (Peterson et al 2004). In fact the concatenated dataset analysis also strongly supports the topology derived from the CAL sequences, which is the most parsimonious phylogeny in terms of intron loss and gain. Although intron sequences were searched against GenBank with BLAST to look for possible functions of the DNA, none were found. Lack of function is consistent with the presence or absence of introns in the examined species. Four of the species have no EF-1 introns, two species have the intron at codon 26, and two of the species have a previously unknown intron at codon 20. If there were any function assignable to the introns, greater conservation of those introns would be expected. The lack of introns in some species does not appear to reduce fitness in the species and thus supports the idea of nonfunctionality.

	ACKNOWLEDGMENTS

 
SWP acknowledges the skillful technical assistance of Jennifer J Scoby in all phases of the systematics study and the SEM operator Arthur J Thomson. FEV thanks Ann Sidor for field assistance in Hawaii and for technical work in the laboratory. MH Hertwig-Jaksch translated the diagnosis into Latin. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.

	FOOTNOTES

 
Accepted for publication February 17, 2005.

¹ Corresponding author. E-mail: peterssw{at}ncaur.usda.gov

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