Reclassification of an isolate of Guignardia citricarpa from New Zealand as Guignardia mangiferae by sequence analysis
Abstract
Citrus black spot was recorded as present in New Zealand in international databases on the basis of one isolate (ICMP 8336) identified by morphological features as Guignardia citricarpa. This isolate was from a soft rot, not a typical symptom of citrus black spot, on a Seminole tangerine fruit grown in the northern region of New Zealand. Sequence analysis of the internal transcribed spacer (ITS) region (ITS1, 5·8S and ITS2) showed that this isolate was 99% identical to the ITS region of G. mangiferae, a closely related saprotroph. Despite climatic conditions being suitable for this disease, citrus black spot symptoms have never been seen on citrus fruits grown in New Zealand. Thus the absence of symptoms on citrus in New Zealand is probably because G. citricarpa is not present. On the basis of these results, the record of citrus black spot occurring in New Zealand should be re-examined.
Introduction
Citrus black spot is a fungal disease caused by Guignardia citricarpa, first reported in Australia by Benson (1895) and later named and described by Kiely (1948). Citrus black spot has since been recorded in Russia, Asia (Bhutan, China, Indonesia and the Philippines), Africa (Kenya, Mozambique, South Africa, Zambia and Zimbabwe), South America (Argentina and Brazil) and Oceania (Australia, New Zealand and Vanuatu) (CAB International, 2000).
Two strains deemed to be G. citricarpa were isolated from citrus by McOnie (1964). The pathogenic strain produced symptoms on inoculated orange fruits and grapefruits, while the other, saprotrophic strain did not. These two strains could also be distinguished on the basis of morphological features. That is, all isolates of the pathogen produced pycnidia in culture and on old, dead citrus leaves, but pycnidia were not produced by saprotrophic isolates. Instead, all saprotrophic strains produced mature perithecia. Although some pathogenic isolates produced perithecia, these did not form asci. Also, the saprotrophic strain grew twice as quickly as the pathogenic strain on all artificial media tested.
Citrus black spot can cause significant economic losses by disfiguring the fruits, which are then rejected for export, and in severe outbreaks it causes fruit drop in the orchard. All citrus except sour orange (Citrus aurantium) are susceptible. Symptoms described during an epidemic in Australia were of round, dark, depressed spots occurring in three stages of increasing severity: hard spot, freckle spot and virulent spot (Kiely, 1948). Hard spot symptoms consist of lesions several mm in diameter with a green border, which appear on mature fruits in early spring, but do not develop further. Freckle spot symptoms develop on fruits in 2–4 days on the side exposed to the sun, and are orange to brick-red, turning brown, without a green border. In 2–3 weeks of warm temperatures, individual freckle spots can merge to cover up to two-thirds of the fruit, resulting in the virulent spot symptom. The latter can also develop from previously symptomless fruit tissue.
Symptoms of citrus black spot have never been recorded on citrus fruits grown in New Zealand, either in an official survey a decade ago (MAF, 1992) or since. The record of occurrence in New Zealand is based on one isolate from Seminole tangelo (Citrus paradisi × C. reticulata) from symptoms that were not typical of black spot. This isolate was described as possibly pathogenic because it produced pycnidia freely in culture (C.F. Hill, Ministry of Agriculture and Fisheries (MAF), National Plant Pest Reference Laboratory, Tamaki, Auckland, New Zealand, personal communication). Pathogenicity on citrus was not tested. The identity of this isolate was confirmed as G. citricarpa on the basis of morphological features by Dr A. Sivanesan (International Mycological Institute, IMI).
Morphological features were later found to be unreliable for distinguishing the saprotrophic from the pathogenic strains of G. citricarpa. First, a number of isolates of G. citricarpa were shown to produce both pycnidia and ascocarps (Frean, 1966; Wang & Tsai, 1974; Moran Lemir et al., 2000) and thus could not be separated using this morphological feature. Second, Baayen et al. (2002) showed overlap in growth rates between isolates of the pathogenic and saprotrophic strains, which reduced the usefulness of this criterion for separating strains.
Because of the unreliability of morphological features for identification, later workers investigated the use of sequence information for distinguishing pathogenic from saprotrophic strains. In these studies the pathogenic isolates were those from fruits with symptoms of citrus black spot, and the saprotrophic isolates were from symptomless fruits or leaves, or from small spots on fruits not typical of citrus black spot. Meyer et al. (2001) and Baayen et al. (2002) were able to reliably separate a large number of isolates of pathogenic and saprotrophic strains on the basis of ribosomal DNA internal transcribed spacer (ITS) sequence. Examination of a few isolates showed that the thickness of the mucoid sheath of conidia was a reliable morphological feature that could be used to separate strains, but more isolates need to be examined before this method can be used (Baayen et al., 2002).
On the basis of ITS sequence, morphological comparisons between the two strains of G. citricarpa and a description by Roy (1968), Baayen et al. (2002) named the saprotrophic strain Guignardia mangiferae A.J. Roy. The name G. citricarpa now refers to the pathogenic strain only, which causes the disease citrus black spot.
The isolate identified as G. citricarpa on which the New Zealand record is based (CMI, 1990) is from a Seminole tangelo fruit sent to the MAF Auckland Diagnostic Station from one of the major citrus-growing regions in New Zealand (Kerikeri, 35°S, 174°E) in August 1983. This isolate was deposited in two collections: the International Collection of Micro-organisms from Plants (ICMP) (Manaaki Whenua Landcare Research, Auckland, New Zealand) as ICMP 8336, and the IMI Herbarium (Bakeham Lane, Egham, UK) as IMI 280084 and IMI 280085, in September 1983.
Because identification of ICMP 8336 was based on morphological features alone, and the citrus black spot disease is not present in New Zealand, it is possible that this isolate is not G. citricarpa. ITS sequencing has since been shown to be a reliable method of distinguishing G. citricarpa and G. mangiferae, and this technique was used to determine the true identity of ICMP 8336.
Materials and methods
Fungi used in molecular studies
Five cultures deposited as G. citricarpa were obtained from ICMP (Table 1).
| Species | ICMP number | Other numbers | Host isolated from | Black spot symptoms | Depositor(s) | Country of origin | Year of isolation | Reference |
|---|---|---|---|---|---|---|---|---|
| G. citricarpa | 7825 | Citrus sp. | Probably | G. Laundon | Port intercept, New Zealand | 1982 | Young & Fletcher (1997) | |
| G. citricarpa | 7872 | 32 757a | Citrus lemon | No | C.S. Huang | Taiwan | 1974 | Wang & Tsai (1974) |
| C.K. Wang | ||||||||
| G. citricarpa | 7873 | 26 254a | Valencia orange | Yes | H.T. Brodrick | South Africa | 1970 | Brodrick & Rabie (1970) |
| G. citricarpa | 7874 | 32 758a | Mandarin orange | Yes | C.S. Huang | Taiwan | 1974 | Wang & Tsai (1974) |
| C.K. Wang | ||||||||
| G. citricarpa | 8336 | 280 084b | Seminole tangarine | No | C.F. Hill | New Zealand | 1983 | Young & Fletcher (1997) |
| 280 085b |
- a American Type Culture Collection.
- b IMI Herbarium, Bakeham Lane, Egham, UK.
Fungal DNA extraction
Fungal spores and mycelium were harvested from 12-week cultures grown on potato dextrose agar (Difco) by adding 1–2 mL sterile deionized water (SDW) and scraping the surface with a glass rod. The resultant slurry was removed and DNA was extracted using a modified method originally developed for rust fungi (Pei & Ruiz, 2000). In this method the washed mycelial and spore slurry was disrupted by grinding, then resuspended in extraction buffer (150 mm EDTA pH 8, 5 mm Tris pH 8, 1% SDS) and treated with RNase (10 µg). Finally, DNA was extracted with phenol : chloroform : iso-amyl alcohol (1 : 0·96 : 0·28 v/v) and ethanol-precipitated. Otherwise, DNA was extracted using the DNeasy Plant Mini Kit (Qiagen).
ITS sequencing
The ITS region was amplified using the primer set ITS1/ITS4 (White et al., 1990) under the following reaction conditions. The PCR reaction mixture (25 µL final volume) contained c. 5 ng template DNA, 0·5 µm of each primer, 400 µm of each dNTP, 1 U Platinum Taq DNA Polymerase (Invitrogen), Taq DNA polymerase buffer and 3 mm MgCl2. The reaction mixture was placed in a thermocycler (Techne) for an initial denaturation of 95°C for 4 min, followed by 35 cycles at 95°C for 30 s (denaturing), 55°C for 30 s (annealing) and 72°C for 45 s (elongation). A final elongation step of 10 min at 72°C was carried out. The PCR products were then purified by gel electrophoresis (10 g agarose L−1) followed by treatment with the Molecular Biochemicals High Pure PCR Product Purification Kit (Roche Diagnostics). Purified PCR products were sent to the University of Waikato, Hamilton, New Zealand for sequencing reactions. DNA templates for sequencing were prepared using DYEnamicET dye terminator chemistry and ITS 1 and 4 primers. DNA sequences were resolved using a MegaBACE 500 DNA analysis system fitted with 40-cm capillary arrays (Amersham Pharmacia Biotech) loaded with linear polyacrylamide long-read matrix. Sequences were analysed using blast (National Center for Biotechnology Information; NCBI) and vector nti 8 (InforMax Inc.) and compared with representative ITS sequences from GenBank (NCBI) for G. mangiferae and G. citricarpa (Table 2). Because there was a higher percentage of polymorphisms in ITS1, a section of this region was selected to confirm identity for ICMP isolates 7825, 7872, 7873, 7874 and 8336. The region of ITS1 from base 7 to base 74 was selected for this purpose.
| GenBank accession number | Fungus | Isolate number | Sequence | Length | Reference |
|---|---|---|---|---|---|
| AF346781 | G. citricarpa | Clone 68 | partial 18S ribosomal RNA gene, ITS1, 5·8S ribosomal RNA gene, ITS2, partial 28S ribosomal RNA gene | 592 | Meyer et al. (2001) |
| AY042921 | G. citricarpa | GC1 | ITS1 | 234 | Baayen et al. (2002) |
| AY042922 | G. citricarpa | GC1 | ITS2 | 226 | Baayen et al. (2002) |
| AY042915 | G. mangiferae | GC10 | ITS1 | 231 | Baayen et al. (2002) |
| AY042916 | G. mangiferae | GC10 | ITS2 | 223 | Baayen et al. (2002) |
| AY277709 | G. mangiferae | 99-37 | partial 18S ribosomal RNA gene, ITS1, 5·8S ribosomal RNA gene, ITS2, partial 28S ribosomal RNA gene | 624 | Rodrigues et al. (2004) |
| AY816311 | G. mangiferae | 8336 | partial 18S ribosomal RNA gene, ITS1, 5·8S ribosomal RNA gene, ITS2, partial 28S ribosomal RNA gene | 632 | This study |
Results
Sequence analysis of ITS1/ITS4 primer product
A sequence of 632 bp was obtained for ICMP 8336 following PCR using the ITS1/ITS4 primer set and deposited as AY816311 in GenBank. Comparison with published sequences using blast showed that this sequence comprised 29 bp of 18S ribosomal RNA gene, ITS1; 5·8S ribosomal RNA gene, ITS2; and 52 bp of 28S ribosomal RNA sequence. In this region, ICMP 8336 was 99% identical to G. mangiferae AY277709 when comparing all 624 available base pairs. There were no gaps, and 623 of 624 bases were identical. Comparison with G. citricarpa AF346781 showed that 487 of 534 bases (91%) were identical, with six gaps.
There were significant differences from the sequences of G. citricarpa in both ITS1 and ITS2 (Fig. 1). ICMP 8336 was dissimilar to G. citricarpa in 48 of the 243 bases (19·8%) of ITS1 and in 14 of 126 bases (11·1%) in the ITS2 region. In contrast, ICMP 8336 was 100% similar to G. mangiferae AY277709 in ITS1, and 121 of 122 bases (98·4%) were similar in ITS2.
Sequence alignment of rDNA of products obtained using ITS primers 1 and 4 of ICMP isolate 8336 compared with NCBI sequence of AY277709 (Rodrigues et al., 2004), AY043915 and AY042916 (Baayen et al., 2002) (Guignardia mangiferae); and AF346781 (Meyer et al., 2001), AY042921 and AY042922 (Baayen et al., 2002) (G. citricarpa). Positions of primers ITS1 and ITS4, are indicated. –, Gaps generated by vector nti 8 during alignment. Lower-case letters, ITS4 primer sequence. Positions of 18SRNA, ITS1, 5·8S RNA, ITS2 and 28SRNA are from G. mangiferae AY277709.
Sequence comparisons of region of polymorphism in ITS1
Sequence data are presented for the region of polymorphisms in ITS1 from bases 7 to 67 (Fig. 2). Comparison of this region of polymorphism with published sequence data using blast analysis confirmed the identity of ICMP 7825, 7873 and 7874 as G. citricarpa (100% similarity), but ICMP 7872 and 8336 were reclassified as G. mangiferae (100% similarity) (Table 3).
Alignment of the region of polymorphism in ITS1 from bases 37–103. ICMP 7872 and 8336 are 100% identical with G. mangiferae AY042915 (Baayen et al., 2002); ICMP 7825, 7873 and 7874 are 100% identical with G. citricarpa AY042921 (Baayen et al., 2002).
| Species | ICMP number | Other numbers | Reference |
|---|---|---|---|
| G. citricarpa | 7825 | This study | |
| G. mangiferae | 7872 | 32 757a | Baayen et al. (2002) and this study |
| G. citricarpa | 7873 | 26 254a | This study |
| G. citricarpa | 7874 | 32 758a | This study |
| G. mangiferae | 8336 | 280 084b | This study |
| 280 085b |
- a American Type Culture Collection.
- b IMI Herbarium, Bakeham Lane, Egham, UK.
Discussion
Sequencing the rRNA 18S 23S intertranscribed spacer region has allowed the reclassification of species of Guignardia that had been wrongly identified before their deposition in culture collections. This concerns mainly G. mangiferae (ICMP 8336). This isolate was from a fruit rot on a Seminole tangelo that was sent from Kerikeri to a MAF laboratory in Auckland, New Zealand in August 1983. In September 1983 it was deposited in two culture collections, the ICMP collection of Landcare Research New Zealand, and the International Mycological Institute. It was then identified as G. citricarpa on the basis of morphological features. Subsequent studies showed that only ITS sequence was reliable for identifying G. citricarpa (Meyer et al., 2001; Baayen et al., 2002), and sequence analysis of ITS regions of ICMP 8336 resulted in reclassification of this isolate as G. mangiferae, a closely related saprotroph.
ICMP 7872 was also incorrectly identified as G. citricarpa in the ICMP collection and in its original deposition in the American Type Culture Collection. Baayen et al. (2002) also sequenced the ITS region of this isolate (ATCC 32757), and confirmed its identity as G. mangiferae. Consistent with its identification by sequence as the saprotrophic strain (G. mangiferae), this culture had a faster growth rate than other isolates tested, and was from a lemon leaf rather than from fruit (Wang & Tsai, 1974). However, both pycnidia and perithecia were produced in culture.
Sequence comparison of a short piece of the ITS1 rDNA containing a high proportion of polymorphisms proved reliable for distinguishing G. citricarpa from G. mangiferae. This short segment of ITS1 sequence could be obtained in 2–3 days without the more intense sequencing effort required for the entire 632 bp amplified by conventional ITS primers. Primers could be designed specifically to amplify this section of rDNA.
The climatic conditions in New Zealand are suitable for citrus black spot on the basis of laboratory temperature and wetness studies. For instance, Lee & Huang (1973) showed that temperatures of 21–28°C are necessary for ascospore formation and discharge. These temperatures are within the range of mean daily maximum temperatures that occur in northern citrus-producing regions of New Zealand (e.g. Kerikeri) during the summer months of November–February (Moir et al., 1986). Also required were alternating periods of wet and dry (Lee & Huang, 1973). Rainfall is relatively high throughout the year in all regions in New Zealand, and during November–March mean monthly rainfall is c. 100 mm in the northern citrus-growing region (Moir et al., 1986). In South Africa, also in the southern hemisphere, the period of susceptibility of fruit to infection is from November to January, during early fruit set (Kotzé, 1981). Fruit set also occurs in New Zealand during this period, and temperatures are most suitable for infection and spread by citrus black spot at this time. Because the climate is suitable for establishment and spread of citrus black spot disease, it is important now to include G. citricarpa on the unwanted organisms list for New Zealand.
McOnie (1964) found both G. citricarpa and G. mangiferae present in the same citrus orchards in South Africa. This suggests that climatic conditions suitable for G. mangiferae are also suitable for G. citricarpa. The isolation of G. mangiferae ICMP 8336 from a citrus fruit in New Zealand provides further evidence for the climatic suitability of New Zealand for citrus black spot.
The evidence provided here for the identity of ICMP 8336 as G. mangiferae, based on sequence, suggests that the one record on which the presence of G. citricarpa in New Zealand is based is incorrect. The lack of observed disease symptoms is probably because the fungus G. citricarpa is not present in New Zealand. In conclusion, New Zealand should be reclassified as being free of citrus black spot.
Acknowledgements
This study was supported by a grant from the Foundation for Research Science and Technology, contract no. CO6X0217. We are grateful to Joy Tyson and Robert Taylor for valuable discussion, and to Joy Tyson for access to e-mail correspondence with IMI and to an unpublished report to MAF.
