Sequence diversity of β-tubulin (tubA) gene in Phaeosphaeria nodorum and P. avenaria


  • Edited by B. Paul

*Corresponding author. Tel.: +1 301 504 6308; fax: +1 301 504 5449., E-mail address:


Full-length coding sequences of the β-tubulin gene (tubA) were PCR-amplified and sequenced from 42 Phaeosphaeria isolates, including 16 P. nodorum and 23 P. avenaria species from cereals, two Polish isolates from rye (Secale cereale L.), and one isolate from dallis grass (Paspalum dilatatum Poir). A tubA gene of size 1556 bp was identified in wheat- and barley-biotype P. nodorum (PN-w and PN-b), P. avenaria f. sp. avenaria (Paa), homothallic P. avenaria f. sp. triticea (Pat.) (Pat1) and the Pat. isolate (Pat3) from the State of Washington. The tubA gene length polymorphisms were detected in two Pat. isolates (Pat2) from foxtail barley (Hordeum jubatum L.), one from dallis grass and two Polish isolates from rye. These size differences were due to the variation of intron lengths among these three Phaeosphaeria species. All Phaeosphaeria isolates have identical 1344 bp exons that can be translated into a 447 amino acid β-tubulin. Like glyceraldehyde-3-phosphate dehydrogenase, the β-tubulin amino acid sequence was identical in all Phaeosphaeria species used in this study, with the exception of the two Pat2 isolates. Six amino acid differences were evident in the β-tubulin of these Pat2 isolates.


β-tubulin is one of the important components of microtubules, which are essential for cellular structures, such as mitotic spindles, the cytoskeleton and axonemes in eukaryotic cells. The tub genes are highly conserved in eukaryotes, and molecular analysis of these tub genes is widely used to identify and study phylogenetic relationships among fungi [1–3]. Ascomycetes contain one or more β-tubulin (tub) genes [4,5]. The lack of a particular intron, the difference in numbers of gene and the diverse DNA and deduced peptide sequences of the tub genes have been used to distinguish and assess the relationship among different species and within a single species of fungi [6–11]. Mutations in the tub gene confer benomyl tolerance in some fungi and have been used as a dominant selectable marker in fungal transformation [9,12,13].

The Septoria blotch disease is a disease complex caused by a number of fungi that are not always closely related [14,15]. The four pathogens of the Septoria group that have had the greatest impact on overall agriculture in cereals are Mycosphaerella graminicola (anamorph, Septoria tritici), Septoria passerinii, Phaeosphaeria nodorum and P. avenaria[16–20]. Identification of two important Phaeosphaeria pathogens, P. nodorum (E. Müller) Hedjaroude [/Leptosphaeria nodorum E. Müller], anamorph: Stagonospora nodorum (Berk.) E. Castellani et E.G. Germano. [/Septoria nodorum (Berk.) Berk. in Berk. & Broome] and P. avenaria (G.F. Weber) O. Eriksson [/Leptosphaeria avenaria G.F. Weber], anamorph: Stagonospora avenae (A.B. Frank) Bissett [/Septoria avenae A.B. Frank] is largely based on the pycnidiospore morphology of their anamorph stages [18–21]. Based on its pathogenicity to wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.), P. nodorum was further recognized as two formae speciales, a wheat-biotype (PN-w) and a barley-biotype (PN-b) [22,23]. P. avenaria was also divided to two formae speciales; P. avenaria f. sp. avenaria (Paa), infecting oats (Avena spp.), and P. avenaria f. sp. triticea (Pat) from wheat, barley, rye (Secale cereale L.) or several grasses [24–26].

In recent years, genetic relatedness and differentiation of cereal Phaeosphaeria species have been re-examined at the molecular level with molecular data. Restriction fragment length polymorphism (RFLP) fingerprinting as well as sequence data from the rDNA internal transcribed spacer (ITS) and mating type gene conserved region have been used to define genetic relationships among the wheat- and barley-biotypes in P. nodorum (PN-w and PN-b), three genetically distinct groups (Pat1, Pat2 and Pat3) of P. avenaria f. sp. triticea (Pat), and P. avenaria f. sp. avenaria (Paa) [27–30]. These molecular data indicated that Paa is more closely related to PN-b, homothallic Pat1, and Pat3 than Pat2 and PN-w. With the exception of Pat2, the deduced amino acid sequences of glyceraldehyde-3-phosphate dehydrogenase (GPD) (EC1.2.1.12) protein are identical in all Phaeosphaeria species studied [31]. However, analysis of the glyceraldehyde-3-phosphate dehydrogenase (gpd) nucleotide sequences strongly supports the defined phylogenetic relationships among Phaeosphaeria species [31].

Development of a reliable method for Phaeosphaeria species identification will help to diagnose cereal Septoria diseases and to identify for potential disease control strategies. The results based on molecular methods will also facilitate the development of a phylogenetic hypothesis for deep branches within the Kingdom of Fungi and enhance research and educational tools in fungal systematics. In order to provide molecular evidence for supporting species designations and their evolution in cereal and other Phaeosphaeria species, the present study was to examine the sequence variations of highly conserved tubA gene.

2Materials and methods

Phaeosphaeria pathogens were either collected from Canada, Poland and USA between 1975 and 2002 or purchased from American Type Culture Collection (ATCC, Manassas, VA) (Table 1). Procedures for maintaining and growing fungal cultures were previously described [30]. Genomic DNA from 11 wheat- (PN-w) and 5 barley-biotype P. nodorum (PN-b) isolates, 15 P. avenaria f. sp. triticea (Pat.) isolates including 12 homothallic Pat1, 2 Pat2 from foxtail barley (Hordeum jubatum L.) and a Pat3 from the State of Washington, 8 P. avenaria f. sp. avenaria (Paa) isolates, a Phaeosphaeria isolate (S-93–48) from dallis grass (Paspalum dilatatum Poir.), and two Phaeosphaeria rye isolates (Sn23-1, Sn48-1) from Poland, were used as templates for PCR amplification [31,32]. Five primer pairs, 2A/2B (nt542-563/nt1032-1009; 5′-GTGCATCTGTAGCTGCCTGTTG/5′-GGAGATCCGAAGTGCCATTGTAGA), 1A/1B (nt617-638/nt1259-1237; 5′-CAAGCGTTCTCACCGACTTGTC/5′-CAGTTGTTACCAGCACCAGACTG), 6A/6B (nt1009-1032/nt1593-1573; 5′-TCTACAATGGCACTTCGGATCTC/5′-CATGCAAATGTCGTAGAGAGC), 7A/7B (nt1237-1259/nt1986-1965; 5′-CAGTCTGGTGCTGGTAACAACTG/5′-CTCAACGAAGTAGGACGAGTTC) and 3A/3B (nt1848-1871/nt2668-2648; 5′-GAACATGATGGCTGCCTCTGACTT/5′-AGCTGGGACTGCGGTATCTTT), designed from the tubAR gene of P. nodorum isolate BSm300 (Accession number S56922) were used to amplify the corresponding full-length gene in other Phaeosphaeria species [33]. Instead of the primer pair 6A/6B, primer pair 6E/6F (nt1050-1073/nt1472-1450; 5′-GTCTACTTCAACGAGGTGCGTATC/5′-GAAGGCACAACGGAGAAAGTGGC) was used for gene amplification in the Pat3 isolate S-81-W10. PCR amplification was performed in 50 μl reaction mixtures containing 1× reaction buffer (50 mM KCl, 10 mM Tris–HCl, pH 9.0 (25°C), 0.1% Triton X-100), 1.25 mM MgCl2, 0.2 mM dNTPs, 2 μM each primer, 80 ng genomic DNA, and 1.0 unit of Taq DNA polymerase (Promega, Madison, WI). Reaction parameters were: denaturation (94°C, 3 min) followed by 40 cycles of 94°C (20 s), 55°C (30 s), and 72°C (1 min), and a final incubation at 72°C (10 min). Isolation and direct sequencing of PCR products were conducted as described previously [31].

Table 1.  Isolates of Phaeosphaeria species used in β-tubulin gene analysis
SpeciesOriginal hostYearGeographic locationGenBank accession number
Phaeosphaeria nodorum (wheat-biotype) (PN-w)
9074Wheat (Triticum aestivum L.)1983Gallatin County, MT, USAAY786339
9076Wheat1986Richland County, MT, USA(=AY786339)
8408Wheat1986Mandan, ND, USA(=AY786336)
9506Wheat1987Mandan, ND, USA(=AY786336)
S-74–20AWheat1975Griffin, GA, USAAY786334
S-80–301Triticale (xTriticosecale)1980Bledsoe, GA, USAAY786335
S-81-B13BBarley1981Bledsoe, GA, USA(=AY786337)
Sn26–1WheatRzeszów, PolandAY786338
Sn27–1WheatSieradz, PolandAY786336
Sn37–1WheatSzelejewo, PolandAY786337
98–12981Rye (Secale cereale L.)1998Mandan, ND, USA(=AY786336)
Phaeosphaeria nodorum (barley-biotype) (PN-b)
S-82–13Barley (Hordeum vulgare L.)1982Senoia, GA, USAAY786332
S-83–2Barley1983Tifton, GA, USA(=AY786332)
S-83–7Barley1983Holland, VA, USA(=AY786332)
S-84–2Barley1984Moultrie, GA, USA(=AY786332)
S-92–7Barley1992Raleigh, NC, USA(=AY786332)
Phaeosphaeria avenaria f. sp. triticea (Pat1)
10052–2Wheat1988Langdon, ND, USA(=AY786329)
12618Wheat1995Dickinson, ND, USA(=AY786329)
12889Wheat1997Mandan, ND, USAAY786329
13050–2Barley1998Dunn County, ND, USA(=AY786329)
13061Barley1998Morton County, ND, USA(=AY786329)
13077–2Barley1998Towner County, ND, USA(=AY786329)
Sa38–1Oat2001Radzików, Poland(=AY786329)
Sa39–2Oat2001Radzików, Poland(=AY786329)
Sat22–2Rye1995Podkarpackie, Poland(=AY786329)
Sat23–2Triticale1995Mazowieckie, Poland(=AY786329)
Sat23–8Triticale1995Mazowieckie, Poland(=AY786329)
Sat24–1WheatWarmińsko-Mazurskie, Poland(=AY786329)
Phaeosphaeria avenaria f. sp. triticea (Pat2)
ATCC26370Foxtail barley (Hordeum jubatum L.)Minnesota, USAAY786330
ATCC26377Foxtail barleyMinnesota, USA(=AY786330)
Phaeosphaeria avenaria f. sp. triticea (Pat3)
S-81-W10Wheat1981Washington, USAAY823527
Phaeosphaeria avenaria f. sp. avenaria (Paa)
5413Oat1983Ontario, CanadaAY870401
1919WRSOat2002Manitoba, CanadaAY870402
1920WRSOat2002Manitoba, Canada(=AY786327)
1921WRSOat2002Manitoba, CanadaAY870403
ATCC12277Oat (Avena sativa L.)USAAY786327
Sa37–2Oat2001Radzików, PolandAY870404
SAA001NY–85Oat1985New York, USAAY786328
SAT002NY–84Wheat1984New York, USAAY870405
Phaeosphaeria sp.
Sn48–1Winter rye1995Jelenia Góra, PolandAY786331
Sn23–1Winter ryeBydgoszcz, Poland(=AY786331)
S-93–48Dallis grass (Paspalum dilatatum Poir.)1993Griffin, GA, USAAY786333

Phylogenetic relationships among cereal Phaeosphaeria species were analyzed using the full-length tubA gene sequences. Correlation of cereal Phaeosphaeria with other fungi belonging to Order Dothideales, Venturia, Leptosphaeria and Mycosphaerella, was studied with β-tubulin amino acid sequences. The full-length amino acid sequences were retrieved from the GenBank ( The DNA and peptide sequences were aligned with ClustalX (1.83) in a multiple sequence alignment mode [34]. From the aligned sequences, 500 data sets were generated by bootstrap re-sampling using the ‘seqboot’ program of Phylogeny Inference Package (Phylip) Version 3.6 (alpha2) (Felsenstein 2004; The bootstrapped data sets were evaluated by the maximum likelihood (ML) method using the ‘dnaml' program for DNAs and ‘proml' for peptides. Finally, the ‘consense’ program was used to construct a ‘tree’[35].

3Results and discussion

3.1Structure of the tubA gene

The tubA gene coding sequence amplified from Phaeosphaeria species differed in length (Table 2). The tubA gene from wheat- and barley-biotypes of P. nodorum (PN-w and PN-b), P. avenaria f. sp. avenaria (Paa), homothallic P. avenaria f. sp. triticea (Pat1) and the Pat3 isolate from the State of Washington had the same length, 1556 bp, while the same gene from Pat2 isolates and the Phaeosphaeria spp. from dallis grass was 1554 bp in size. Two Phaeospharia rye isolates from Poland (Sn23-1, Sn48–1) were 1557 bp (Table 2). The tubA gene has three introns and four exons as the tubAR gene of P. nodorum isolate BSm300 (Accession number S56922) [33]. The locations of these three introns among Phaeosphaeria species were highly conserved in other ascomycetes, and the size difference was due to variations in intron 1 and intron 2. In comparison with the tubA gene of P. nodorum, intron 1 of Phaeosphaeria sp. from dallis grass and intron 2 of Pat2 isolates were two bases shorter. However, the two Phaeosphaeria rye isolates (Sn23-1 and Sn48-1) from Poland had one extra base in intron 1.

Table 2.  Structure of the β-tubulin (tubA) gene in Phaeosphaeria species
SpeciesIsolateSize (bp)Intron size (bp)Number of nucleotide substitutionsNumber of amino acid substitutions
  1. aA histidine to tyrosine substitution at position 6 contributes to benomyl resistance.

Phaeosphaeria nodorum (wheat-biotype) (PN-w)Sn27-115561085252
P. nodorumBSm30015561095152202a
P. nodorum (barley-biotype) (PN-b)S-82–1315561085252530
Phaeosphaeria spp. (from rye)Sn48-115571095252180
Phaeosphaeria spp. (From dallis grass)S-93-4815541065252570
P. avenaria f. sp. avenaria (Paa)ATCC1227715561085252560
P. avenaria f. sp. triticea (Pat1)1288915561085252540
P. avenaria f. sp. triticea (Pat2)ATCC2637015541085052686
P. avenaria f. sp. triticea (Pat3)S-81-W1015561085252560

Nucleotide sequence diversity was found in the tubA genes of wheat-biotype P. nodorum (PN-w) and P. avenaria f. sp. avenaria (Paa), but not those from 5 barley-biotype P. nodorum (PN-b) isolates and 12 homothallic isolates of Pat. (Pat1). When compared to the tubAR gene, which is responsible for benomyl-resistance in isolate BSm300 ([33]; Accession number S56922), 6, 18, 20, 21, 21 and 22 nucleotide substitutions were found in S-80-301, S-74-20A, Sn27-1/8408/9506/98-12981, Sn26-1, Sn37-1/S-81-B13B and 9074/9076 isolates, respectively. Our results suggested that isolate S-80–301 is closely related to isolate BSm300 and two Phaeosphaeria rye isolates from Poland (Fig. 1). As compared to the tubA gene in P. avenaria f. sp. avenaria ATCC12277 isolate (Accession number AY786327), the number of nucleotide substitutions in isolate 1920WRS, 1921WRS, SAT002NY-84, SAA001NY-85, 1919WRS, Sa37-2 and 5413 were 0, 2, 2, 4, 4, 8 and 8, respectively. The number of nucleotide differences in the tubA gene between PN-w Sn27-1 isolate (Accession number AY786336) and other Phaeosphaeria species ranged in between 53 and 68 (Table 2). Based on tubA nucleotide sequences, barley-biotype P. nodorum (PN-b) was closely related to Paa and Pat3, and Pat2 isolates from foxtail barley was separated from homothallic Pat1 and wheat-biotype P. nodorum (Fig. 1). These phylogenetic relationships in cereal Phaeosphaeria species are concordant with earlier results based on the RFLP fingerprinting, ITS region of the nuclear rDNA repeats, and the gpd gene [28,31].

Figure 1.

Phylogenetic relationship based on nucleotide sequences of the full-length β-tubulin gene (tubA) in Phaeosphaeria pathogens. GenBank accession numbers are bold, underlined and given in parentheses. Bootstrap values (with 500 replications) of the internal branches are indicated. Sequences from wheat-biotype P. nodorum (PN-w), barley-biotype P. nodorum (PN-b), P. avenaria f. sp. avenaria (Paa), three subgroups (Pat1, Pat2 and Pat3) of P. avenaria f. sp. triticea and Phaeosphaeria species from rye (Sn48-1) and dallis grass (S-93–48) were analyzed.

3.2Deduced amino acid sequence of the tubA gene

The 1344 bp exon sequence of the tubA gene in Phaeosphaeria species encodes a β-tubulin polypeptide containing 447 amino acids. Since most nucleotide differences in the exon sequence occurred at the wobble position of the amino acid-encoding triplets, they do not affect the β-tubulin amino acid sequence. The first position substitution (from C to T) in a leucine triplet at nt885 of the tubA gene in Paa and at nt892 in Pat2 still encoded the same amino acid at aa225 and aa228 in β-tubulin protein, respectively. The sole exception is the amino acid sequence in Pat2. Nucleotide substitutions found in the first (nt775, nt1045, nt1351), second (nt1064, nt1508) and third (nt1506) codon positions of amino acid triplets in the tubA gene resulted in 6 amino acid changes in β-tubulin of Pat2 (Fig. 2). Like the GPD protein, the β-tubulin peptide sequence is identical and well conserved in cereal Phaeosphaeria species and other Phaeosphaeria species used in this study [31]. Amino acid substitutions occurred in GPD and β-tubulin proteins in Pat2 indicated that they might evolve separately from other Phaeosphaeria species.

Figure 2.

Alignment of deduced β-tubulin peptide sequences from cereal Phaeosphaeria species and two Mycosphaerella graminicola isolates. The numbers in parentheses are GenBank accession numbers. ‘.’, Amino acids are the same in reference to the first sequence. The amino acids underlined are those conserved in ascomycetes. The aspargine (N) at aa219, responsible for taxol resistance, is boxed, and the alanine (A) at aa198 in M. graminicola and the tyrosine (Y) at #6 in a P. nodorum mutant for benomyl resistance are shaded. 1 = the Phaeosphaeria species including wheat- and barley-biotype P. nodorum, P. avenaria f. sp. avenaria, two groups (Pat1 and Pat3) of P. avenaria f. sp. triticea and Phaeosphaeria species from rye (Sn23-1 and Sn48-1) and dallis grass (S-93–48).

The β-tubulin is highly conserved in eukaryotes. In comparison with 58 deduced β-tubulins from Ascomycetes, 173 amino acids are conserved in β-tubulin peptide sequence of Phaeosphaeria species (Fig. 2). The tub gene in Mycosphaerella graminicola (anamorph, Septoria tritici), another important causal agent of cereal Septoria disease, is different from Phaeosphaeria species. In the two M. graminicola isolates so far studied, the tub genes have 6 introns, possess 86.8–87.4% identity in exon sequence and 18–22 amino acid substitutions in deduced peptide sequences as compared to P. nodorum isolate Sn27–1 (Accession numbers AJ310917 and AY547264) [36] (Fig. 1). Nevertheless, based on β-tubulin amino acid sequences, M. graminicola and cereal Phaeosphaeria species were grouped together with other fungi from the Order Dothideales (Fig. 3).

Figure 3.

Phylogenetic relationship based on the peptide sequences of β-tubulin in Phaeosphaeria pathogens and other representative ascomycetes. GenBank accession numbers are bold, underlined and given in parentheses. Bootstrap values (with 500 replications) of the internal branches are indicated. *The peptide sequences of Phaeosphaeria pathogens, except that of P. avenaria f. sp. triticea from Minnesota (Pat2) (AY786330), are identical and represented by P. nodorum (AY786334).

3.3Amino acid substitutions related to drug resistance

Amino acid substitutions in β-tubulin can result in temperature sensitivity and drug resistance in eukaryotes. In ascomycetes, the mutated residues of Y422H, T219N or Q/N100I/V in β-tubulin can cause phenotypic changes including cold sensitivity, taxol resistance and rhizoxin resistance, respectively [37–39]. The deduced amino acid sequence of Phaeosphaeriaβ-tubulin had tyrosine (Y) at position 422 and asparagine (N) at positions 100 and 219 suggesting that these isolates may be cold insensitive, rhizoxin sensitive and taxol resistant (Fig. 2). Resistance to benzimidazole fungicides is known to involve positions 6, 50, 134, 165, 167, 198, 200, 241 and 257, in the fungal β-tubulin [40]. The mutated residue of H6Y was found in β-tubulins of benomyl resistant P. nodorum isolate BSm300 and Trichoderma viride mutants [33,41]. However, the mutation of H6Y in β-tubulin does not confer benomyl resistance in a biocontrol fungus Trichoderma virens[42]. In this study, no amino acid substitutions associated with benomyl resistance were found in the Phaeosphaeria species (see Fig. 4).

Figure 4.

Gel electrophoresis of the PCR-amplified partial β-tubulin gene products in four Phaeosphaeria species. Specific primers used for their identification were 5′-GCCACCTCCTCGCAGCAT/5′-CGTGCTGCAATTTTGCCCGCGGA for barley-biotype P. nodorum (PN-b) (A), 5′-GACACCCCCTCGCAGGAT/5′-CGTGCTGCAAATTCGACCACGGG for wheat-biotype P. nodorum (PN-w) (B), 5′-GACAGCCCCCTCGCAGCAT/5′-CGTGCTGCAAATTCGACCACGGG for Phaeosphaeria species from rye (isolate Sn48–1) (C) and 5′-GCCACCTCCTAGCAGCAT/5′-CGTTCTGCAAAATTGACCGGGGA for Phaeosphaeria species from dallis grass (isolate S-93-48) (D). ‘M’ was 123 bp ladder marker and ‘1-8’ indicated Paa (isolate ATCC12277), Pat1 (isolate 12889), Pat2 (isolate ATCC26370), Pat3 (isolate S-81-W10), PN-b (isolate S-84-2), PN-w (isolate Sn26-1), Sn48-1 and S-93-48, respectively.

3.4Identification of Phaeosphaeria spp. with PCR-based methods

Recently, PCR-based methods have been used to distinguish plant pathogens in a mixed infection and to detect fungicide resistance and genetic variation in a pathogen population [43–46]. Fraaije et al. [36] have designed tub gene specific primers that can be used to detect and quantify Septoria tritici, P. nodorum and two rust fungi on diseased wheat. The specific primer set SNSP7/CONS1 selected for P. nodorum detection amplifies an identical 464 bp size band in all Phaeosphaeria species used in this study, and thus, was not directly suitable for their differentiation (Ueng P.P., unpublished data). Because the tubA gene sequence was highly conserved, it was difficult to develop a PCR amplification method for fast, clear and direct detection of all cereal Phaeosphaeria pathogens. With the PCR reaction parameters described in Section 2 and an annealing temperature at 65°C, primer sets designed from the diverse intron 1 and intron 3 sequences of the tubA gene (nt22–39/nt350–328) could be used to detect wheat-biotype P. nodorum (PN-w), two Phaeosphaeria rye isolates from Poland and the Phaeosphaeria species from dallis grass by amplifying a 329 bp size fragment (Fig. 4B–D). However, one successful way to differentiate these Phaeosphaeria species was using the specific enzymatic restrictions and gel electrophoresis fingerprinting of 1A/1B primer set-mediated amplification products (Table 3). The endonuclease restriction sites were deduced from the nucleotide sequence data and were experimentally demonstrated by enzymatic restrictions and agarose gel electrophoresis (Ueng P.P., unpublished data).

Table 3.  Comparison of endonuclease restriction of PCR-amplified β-tubulin (tubA) gene products in Phaeosphaeria leaf pathogens
Endonuclease enzymesPaaPat (Pat1)Pat (Pat2)Pat (Pat3)Pn (Wheat-biotype)Pn (Barley-biotype)P. spp. (Sn48–1)P. spp. (S-93–48)
646 bp646 bp647 bp646 bp644 bp646 bp644 bp644 bp 
  1. Fragment sizes were given in base pairs (bp). ‘–’ not cut by endonuclease enzymes. 1A/1B primers were used to amplify the fragment from Paa (P. avenaria f. sp. avenaria), Pat (P. avenaria f. sp. triticea), Pn (P. nodorum) and two other Phaeosphaeria species.

Dde I69, 578299, 345
Hin fI237, 409237, 409237, 409
Kpn I87, 55987, 559
Nci I93, 55393, 55393, 55393, 55193, 55393, 551
Pst I178, 468176, 468178, 468176, 468178, 466
Sac II183, 463-183, 463183, 463
Sal I281, 365284, 363
Sph I157, 489157, 487
Stu I142, 505


We thank Rosemarie Hammond and Robert Owens of the USDA-ARS for reviewing the manuscript.