Morphological and molecular characterization of Fusarium spp ...

J Gen Plant Pathol (2012) 78:160–169 DOI 10.1007/s10327-012-0379-5

FUNGAL DISEASES

Morphological and molecular characterization of Fusarium spp. associated with yellowing disease of black pepper (Piper nigrum L.) in Malaysia Sahar Shahnazi • Sariah Meon • Ganesan Vadamalai Khairulmazmi Ahmad • Naghmeh Nejat

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Received: 10 December 2011 / Accepted: 5 March 2012 / Published online: 5 April 2012 Ó The Phytopathological Society of Japan and Springer 2012

Abstract Yellowing disease is one of the most important diseases of black pepper (Piper nigrum L.). To characterize the pathogen(s) responsible for yellowing disease of black pepper in Malaysia, 53 isolates of Fusarium were collected from the roots of diseased black pepper plants and from rhizosphere soils from major growing areas in Sarawak and Johor. A total of 34 isolates of F. solani and 19 isolates of F. proliferatum were obtained and identified based on morphological characteristics and molecular techniques. DNA sequencing of the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA regions was conducted to identify Fusarium species. Nucleotide sequence analysis of the ITS regions revealed that this molecular technique enabled identification of Fusarium at the species level as F. solani and F. proliferatum. In a pathogenicity test on 3-month-old black pepper plants, F. solani was pathogenic, but F. proliferatum was not. On the basis of morphology, DNA sequences and pathogenicity of the fungal isolates from the diseased plants, we showed that yellowing disease on black pepper is caused by F. solani Keywords Fusarium solani Yellowing disease Piper nigrum L. Black pepper Pathogenicity rDNA-ITS S. Shahnazi S. Meon (&) G. Vadamalai N. Nejat Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, D.E., Malaysia e-mail: [email protected] S. Meon G. Vadamalai Department of Plant Protection, Universiti Putra Malaysia, 43400 Serdang, Selangor, D.E., Malaysia K. Ahmad Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia, Campus Bintulu, Sarawak, Malaysia

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Introduction Black pepper (Piper nigrum L.; Piperaceae) is one of the most popular spices in the world. Malaysia is one of the largest producers and exporters of black pepper (Ravindran 2000). However, diseases pose a serious limitation to pepper cultivation, resulting in yield reduction or complete crop loss (Duarte and Albuquerque 1991; Kueh et al. 1993). One of the most important diseases of black pepper is yellowing disease, also known as slow decline (Kueh et al. 1993; Sitepu and Mustika 2000). The symptoms of the disease are yellowing of foliage and root rot, which leads to flaccidity. Once flaccidity has set in, the affected plants die within a short period (Duarte and Albuquerque 1991). During the early stages of pathogen establishment, the growth of young black pepper vines is normal. However, yellowing symptoms appear when the vines are more than 4 months old, measured from the date of field planting. Leaf discolouration usually starts at the bottom of the vines and spreads gradually to the top. In the advanced stages of infection, the plant suffers from defoliation. Yellowing disease may occur both in young and old vines. In serious cases, the main roots lose their feeder roots, resulting in dieback and eventual death of the vines (Kueh et al. 1993; Sitepu and Mustika 2000). Albuquerque (1961) identified the causal agent of yellowing disease as Fusarium solani f. sp. piperis (teleomorph: Nectria haematococca f. sp. piperis) (Hamada et al. 1988). This serious disease causes severe crop losses in most pepper-growing countries, including India, Malaysia, Indonesia, Brazil and Thailand (Duarte and Albuquerque 1991; Ramana and Eapen 2000; Sitepu and Mustika 2000). Fusarium infection on a black pepper plantation is reported to reduce the economic lifespan of the plantation from

J Gen Plant Pathol (2012) 78:160–169

20 years to 6–8 (Duarte and Albuquerque 1991). On a plantation, the affected plants survive for several years, and plant death occurs gradually over 3–4 years (Anandaraj 2000). Accurate and rapid identification of a pathogen is necessary for the appropriate management of the disease (Singh et al. 2006). Morphological characteristics are key for the identification and taxonomy of Fusarium (Leslie and Summerell 2006). The Fusarium taxonomic system has been based on macroconidia and microconidia, morphological characteristics of the chlamydospore, and secondary metabolites (Thrane 1990). Molecular methods, which are faster and more sensitive than morphological identification, are also employed to identify Fusarium species (De Biazio et al. 2008). The use of PCR and DNA sequence analysis of internal transcribed spacer (ITS) regions has become routine for the detection, identification, classification and phylogenetic analysis of many fungi at the species level (Chillali et al. 1998; Henson and French 1993; Hibbett 1992; Oliver 1993; Taylor et al. 2000). ITS regions sequences are highly variable in Fusarium, and taxon-selective ITS amplification has been used to detect fungal pathogens, such as Fusarium (O’Donnell 1992). There is little information about yellowing disease in Malaysia, and Fusarium wilt on black pepper has not been extensively studied in this country. Additionally, the causal fungus of yellowing disease had not been molecularly identified until the present study. The aims of this study were (1) to isolate and characterize Fusarium spp. associated with yellowing disease of black pepper using morphological methods, (2) to identify each Fusarium species using DNA sequence analysis to confirm the results of morphological methods, and (3) to determine the pathogenicity of the Fusarium spp. identified.

Materials and methods Pathogen surveys and fungal isolation Survey sites were chosen based on information from growers about the occurrence of the disease. The roots of black pepper plants with yellowing disease symptoms and rhizosphere soils were collected from major growing areas in Sarawak (Sibu and Sarikei) and Johor (Kulai) in 2009. Fusarium spp. were isolated using either direct root isolation or a soil dilution plate technique on pentachloronitrobenzene (PCNB) medium (Leslie and Summerell 2006). For direct isolation of the Fusarium species from roots, root segments of infected plants were washed to remove excess soil, surface sterilized with 1 % (v/v) sodium hypochlorite solution for 5 min, rinsed twice with sterilized distilled water and allowed to dry on sterilized filter paper.

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After drying, 0.5 cm sections were cut from both ends of the segments, and the centre segments were placed on PCNB media for Fusarium isolation. The plates were incubated at 28 °C for 4–5 days. Fungal colonies growing from the root segments were transferred to fresh potato dextrose agar (PDA). The soil dilution plate technique was performed as described by Parmeter (1970) using PCNB medium. Cultural and morphological characteristics Pure cultures obtained from a single spore of each isolate were grown on PDA to study colony morphology and pigmentation. Carnation leaf agar (CLA) and Spezieller Na¨hrstoffarmer agar (SNA) cultures were used to study the formation and type of macroconidia, microconidia, conidiogenous cells and chlamydospores (Pe´rez-Sierra et al. 2007). PDA plates were incubated at 28 °C in darkness; CLA and SNA plates were incubated at 28 °C with a 12-h photoperiod. All isolates were examined after 10 days using light microscopy and scanning electron microscopy (SEM; JSM-6400, JEOL, Tokyo, Japan). At least 20 microconidia and macroconidia of each isolate were selected randomly for measurement. Samples were prepared for scanning electron microscopy by the modified technique of Al-Awadhi et al. (2002) and Humphreys et al. (1974). Fusarium isolates were identified according to the morphological traits described by Leslie and Summerell (2006). Pathogenicity test Based on cultural characteristics (colony morphology and pigmentation), F. solani isolates were divided into eight groups (Table 1), and F. proliferatum isolates were divided into five groups (Table 2). One isolate from each group was chosen for pathogenicity testing. Three-month-old pepper-plants (cv. Kuching) were uprooted, and the roots were washed under running tap water to remove excess soil. The roots were then wounded with a sterile knife, immersed in a spore suspension (1 9 106 spores/mL) for 5 min, swirled several times and transplanted into pots containing 4 kg sterilized soil mixture (3:2:1 v/v ratio of top soil–peat moss–sand). After transplanting, each pot was inoculated by drenching the soil around the crown with 200 mL of the same spore suspensions (1 9 106 spores/ mL) used for root infection (Edel-Hermann et al. 2011). As a control, roots were wounded and dipped in distilled water, transplanted into pots with sterilized soil and treated with 200 mL sterilized distilled water. A total of 84 plants were used in pathogenicity tests and arranged in a completely randomized design (CRD) with six replications. All pots were maintained in a glasshouse under optimal

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Table 1 Differentiation among Fusarium solani isolates based on colony morphology and pigmentation Group

Isolate

Colony morphology

Pigmentation

Fs-B3

Fs-A3, Fs-B3, Fs-C1, Fs-C2, Fs-D1, Fs-E1, Fs-E3, Fs-F2, Fs-G1, Fs-G3, Fs-H2, Fs-K1, Fs-K7

Delicate, sparse

Light brown

Fs-E2

Fs-A1, Fs-A2, Fs-B1, Fs-B2, Fs-D2, Fs-E2, Fs-F3, Fs-G2, Fs-H3, Fs-K3

Delicate, sparse

Cream

Fs-F5

Fs-F1, Fs-F5

Delicate, sparse

Red

Fs-H4

Fs-H4

Delicate, sparse

Black

Fs-H6

Fs-H6

Floccose

Colorless

Fs-H8

Fs-H5, Fs-H8

Sparse to dense

Cream

Fs-H7

Fs-H7, Fs-H9

Delicate, sparse

Wine

Fs-K6

Fs-K2, Fs-K6

Delicate, sparse

Olive to gray

Isolates in boldface were used in the pathogenicity tests

Table 2 Differentiation among Fusarium proliferatum isolates based on colony morphology and pigmentation Group

Isolate

Colony morphology

Pigmentation

Fp-A1

Fp-A1, Fp-B1, Fp-B2, Fp-D2

Abundant, pannose, white to pale violet, with lobed margin

Dark violet

Fp-F5 Fp-G2

Fp-E3, Fp-F5, Fp-H2, Fp-H8 Fp-E1, Fp-F3, Fp-F4, Fp-G2

Pannose, white to pink Abundant, pannose and white

Pink to light violet No pigment to light pink

Fp-G3

Fp-F1, Fp-G1, Fp-G3

Floccose, white to light purple, growing slower than other isolates and produce rare macroconidia

Dark purple

Fp-H6

Fp-H5, Fp-H6, Fp-H7, Fp-H9

Abundant, pannose, white to light pink

Buff-brown to purple

Isolates in boldface were used in the pathogenicity tests

conditions (32/25 °C day/night cycle), watered daily and assessed regularly. Seven months after inoculation, the percentage of affected plants (plants displaying foliar yellowing and death), the extent of vascular browning, the percentage of root colonisation (Porras et al. 2007) and the effect of infection on the root systems of infected plants (fresh and dry mass of roots) were determined. To determine the amount of colonization of the root by pathogen, root segments of infected plants were randomly sampled, surface sterilized, dried and plated on PCNB medium. Five segments were placed in each plate (4 replicates for each plant). The plates were incubated at 28 °C for 4–5 days. The percentage of root colonization was calculated as the number of root segments with Fusarium divided by the total number of root segments for each plant (Porras et al. 2007). DNA extraction Three to four mycelial plugs from 6-day-old cultures were transferred to 100 mL of potato dextrose broth (PDB) medium and incubated at 28 °C for 6 days. The mycelium was filtered through a double layer of sterile muslin, washed twice with sterile distilled water, drained on filter paper and ground using a mortar and pestle in liquid nitrogen. The genomic DNA of all Fusarium isolates was extracted from 200 mg of ground mycelia using the CTAB

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method (Doyle and Doyle 1990). The quality and concentration of the genomic DNA was assessed using a NanoDrop ND-1000 spectrophotometer (LMS Co., Ltd., Tokyo, Japan), which measured the UV absorbance at 260 and 280 nm and computed the 260/280 absorbance ratio. PCR amplification of ribosomal DNA (rDNA) regions The universal primer pair ITS1–ITS4 was used for amplifying and sequencing the fungal rDNA ITS region (White et al. 1990). The nucleotide sequences were ITS1 (50 -TCC GTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGC TTATTGATATGC-30 ) (First Base Laboratories Sdn. Bhd., Selangor, Malaysia). Amplification reactions were prepared in a total volume of 20 lL containing 2 lL of 109 PCR buffer [100 mM Tris–HCl (pH 8.3), 500 mM KCl, 20 mM MgCl2], 2 lL of dNTP mixture (2.5 mM each), 2 units of Taq DNA polymerase (5 U/lL) (iNtRON Biotechnology, Seoul, Korea), 10 pmol each of forward and reverse primers and 1 lL of template DNA. Thermal cycling consisted of a 2 min initial denaturation at 95 °C, followed by 40 cycles of elongation (denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min), and ending with a final extension at 72 °C for 5 min. A 100-bp ladder (New England Biolabs, Ipswich, MS, USA) was used as a molecular size standard marker. The PCR products were separated by


electrophoresis (at 75 V cm-1 for 50 min) on 1 % (w/v) agarose gels with 19 TBE buffer. The gels were then stained with ethidium bromide to visualize products under UV light using a gel documentation system (Bio-Rad, Philadelphia, PA, USA). Sequence analysis PCR products were purified using a QIAquick gel extraction kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions and sequenced commercially (Medigene Sdn. Bhd., Selangor, Malaysia). All sequences were compared with sequences of Fusarium species available in the GenBank database using BLAST network services for similarities present in the NCBI database (National Center for Biotechnology Information). Multiple sequence alignment was performed using Clustal W (version 1.8) (Thompson et al. 1994). Phylogenetic analyses were constructed by the neighbour joining (NJ) method using MEGA version 4 (Tamura et al. 2007). The bootstrap values illustrated on the phylogenetic dendrogram were generated with 1000 replicate heuristic searches.

Results Identification and morphological characteristics of Fusarium species A total of 53 isolates of Fusarium was obtained from the roots of diseased black pepper plants and from rhizosphere soil. Based on morphological characteristics, 34 isolates were classified as F. solani and 19 isolates as F. proliferatum. Of the 34 isolates of F. solani, 33 isolates were obtained from black pepper roots, except Fs-H8 was isolated from rhizosphere soil, whereas all F. proliferatum isolates were obtained from rhizosphere soil. Our findings fit the description of F. solani (Mart.) Appel & Wollenw. as follows: aerial mycelia were delicate and sparse to dense on PDA. Colonies were cream, white and zonate. Some isolates did not produce pigments (appearing colorless) although some produced cream, red, wine, brown, black or gray pigments (Table 1). Macroconidia were wide, straight to slightly curved, 3- to 5-septate and abundant in sporodochia. The size of macroconidia averaged 23–54 9 4–5 lm. All isolates produced abundant microconidia. Microconidia were oval, ellipsoidal or reniform with 0–1 septum and measured 6–22 9 2–4.5 lm on average (Fig. 1a, b). Conidiophores were long monophialides (Fig. 1f). Sporodochia were cream, blue or green. Chlamydospores formed in the hyphae singly, in pairs or in a short chain. Chlamydospores were globose in shape and smooth- or rough- walled.

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Isolates of F. proliferatum (Mats.) Nirenberg had abundant aerial mycelia that were pannose or floccose and white to light pink on PDA. Pigments in the agar typically varied from light pink or buff to purple, violet or vinaceous (Table 2). Sporodochia were orange, and sclerotia were blue-black (for two isolates). Macroconidia were slender, almost straight to slightly curved, and were typically 3- to 4-septate. The size of macroconidia averaged 23–50 9 2.4–4.0 lm. Microconidia were pyriform, clubshaped with a flattened base, 0- or 1-septate (mostly 0-septate) and found in moderate to long chains with a false head measuring 4–12 9 2–3 lm, on average (Fig. 1c, d). Conidiogenous cells were monophialides and polyphialides (Fig. 1e). Chlamydospores were absent. Pathogenicity tests No symptoms were observed on control plants or plants that were inoculated with F. proliferatum. The symptoms of F. solani infection in the glasshouse-grown plants were yellowing of the foliage and root rot, leading to flaccidity (Fig. 2). All black pepper plants inoculated with F. solani displayed yellowing symptoms 4 months after inoculation. At the end of the experiment (7 months after inoculation), of the 48 plants inoculated with F. solani, only 8.3 % (four plants) displayed flaccidity, and these plants died within 1 week after flaccidity. Vascular browning that extended 26.8 mm (mean) into the stem was observed, the mean percentage of root colonisation was 41.2 %, and the fresh and dry masses of roots were significantly reduced in plants infected by F. solani (Table 3). Isolates of F. solani differed in the percentage of root colonization (Table 4). Fs-F5 was a virulent isolate based on the percentage of root colonization. This isolate produced red pigmentation on culture plates. The pathogenicity test demonstrated that isolates of F. solani (Fs-F5, Fs-H7) that produced red and wine pigments were more pathogenic than other isolates (Table 4). Fusarium solani with the same morphological characteristics as described earlier was reisolated from infected roots, confirming Koch’s postulates. The pathogenicity test suggested that F. solani is the causal pathogen of the disease and that F. proliferatum is present as a saprophyte. Amplification of ITS region On the basis of morphological differentiation and pathogenicity results, 10 isolates of F. solani and 10 isolates of F. proliferatum were selected for molecular identification (Table 5). Amplification of the ITS region of F. solani and F. proliferatum isolates using universal primers ITS1 and ITS4 produced a DNA fragment ca. 550–570 bp long. An identity of 98–100 % was observed between the Fusarium

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Fig. 1 SEM images of macroconidia (a) and microconidia (b) of Fusarium solani, and macroconidia (c) and microconidia (d) of F. proliferatum. Light micrographs of polyphialides (e) of F. proliferatum and monophialide (f) of F. solani

isolates and Fusarium sequences obtained from GenBank. A comparison between sequences available in GenBank and representative isolates from this study indicated that they were similar to several other isolates of F. solani and F. proliferatum originating from different host plants. Therefore, two species, F. solani and F. proliferatum, were identified using ITS region nucleotide sequences. Sequences of representative isolates from both species were deposited in GenBank and used to search for similar sequences in various databanks using the BLAST program (Table 5). A phylogenetic tree inferred from ITS1–5.8S–ITS2 region sequences and generated from the neighbour joining

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(NJ) method illustrated that F. solani and F. proliferatum species clearly form two distinct clades, and this result was supported by a high bootstrap value. Almost no variation was observed among the sequences of F. proliferatum isolates, which clustered in a single group with 100 % homology. Additionally, little variation existed within the ITS region sequences of the F. solani isolates, except for the Fs-H6 isolate (Fig. 3). The ITS1 region had a higher rate of base substitution than in the ITS2 region; the F. solani isolates had a higher rate of base substitution than F. proliferatum isolates in the ITS1 region. Among F. solani isolates, Fs-H6 possessed four nucleotide insertions in the ITS1 sequence, at positions 70 (G),


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Fig. 2 Symptoms of yellowing disease (b–d) in glasshouse. a Healthy plant, b yellowing symptom, c flaccidity and plant death, d root rot

Table 3 Symptoms of black pepper seedlings 7 months after inoculation with Fusarium spp. (mean value for each treatment) Inoculum

Yellowing (%)

Flaccidity and plant death (%)

Root colonization (%)

Extent of browning in stem (mm)

Fresh mass of root (g)

Dry mass of root (g)

F. solani

100

8.3

41.2

26.8

12.8 b

2.4 b

F. proliferatum

_

_

_

_

17.1 a

3.4 a

Control

_

_

_

_

17.3 a

3.6 a

Data were analysed using an ANOVA, and means were compared using Duncan’s multiple range test. Values within a column followed by different letters differed significantly at (P \ 0.05)

71 (A), 117 (A) and 118 (T), and two nucleotide deletions at positions 39 and 129. Additionally, in the ITS2 sequence, Fs-H6 possessed two nucleotide deletions at positions 354 and 371, as measured from the beginning of the ITS1 region, including gaps. Morphologically, isolate Fs-H6 was clearly distinct from other F. solani isolates. Colonies of Fs-H6 were dense and produced no pigmentation.

Discussion One of the most important diseases of black pepper is yellowing disease or slow decline. Yellowing disease is caused by a parasitic fungus (F. solani), although a complex of other biotic and abiotic factors such as parasitic nematodes (Radopholus similis and Meloidogyne incognita), nutrient deficiency, temperature and water stress can

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166 Table 4 Symptoms of black pepper seedlings 7 months after inoculation with Fusarium solani isolates

a

Total number of inoculated seedlings is in parentheses

b

Mean for inoculated seedlings

Table 5 Identification of Fusarium spp. based on ITS region sequencing


Isolate

No. of seedlingsa

Extent of browning in stem (mm)b

Fresh mass of root (g)b

Dry mass of root (g)b

Yellowing

Flaccidity and plant death

Fs-B3

6 (6)

0 (6)

44.9

12.4

14.9

2.5

Fs-E2

6 (6)

0 (6)

32.5

11.3

15.2

2.7

Fs-F5

6 (6)

2 (6)

58.3

74.1

3.5

Fs-H4

6 (6)

0 (6)

53.3

14.8

13.4

2.2

Fs-H6

6 (6)

0 (6)

27.4

10.8

16

2.9

Fs-H8

6 (6)

0 (6)

28.3

11

15.7

2.9

Fs-H7 Fs-K6

6 (6) 6 (6)

2 (6) 0 (6)

56.6 28.3

68.7 11.3

8.1 15.3

1.8 2.8

1

Fusarium species

Isolate code

Location

Accession number

Sequence with best match

F. solani

Fs-B3

Sarawak-Sibu-Field B

JF323005

FJ719812

98

Fs-E2

Sarawak-Sarikei-Field E

JF322996

FJ719812

99

Fs-F5

Sarawak-Sarikei-Field F

JF322997

GQ451337

98

Fs-H4

Sarawak-Sarikei-Field H

JF322998

GQ451337

98

Fs-H5


JF322999

EU625405

100

Fs-H6


JF323000

AF178408

98

Fs-H7


JF323001

FJ719812

98

Fs-H8


JF323002

FJ345352

99

Fs-K2

Johor-Kulai-Field K

JF323003

GQ121291

98

Fs-K6

Johor-Kulai-Field K

JF323004

GQ451337

98

Fp-A1 Fp-B1

Sarawak-Sibu-Field A Sarawak-Sibu-Field B

HQ662410 HQ662411

GU066712 GU066712

100 100

Fp-D2

Sarawak-Sibu-Field D

HQ830365

FJ545380

99

Fp-F3


HQ830366

FJ545380

99

Fp-F5


HQ830367

GU723438

99

Fp-G2

Sarawak-Sarikei-Field G

HQ830368

FJ545380

99

Fp-G3

Sarawak-Sarikei-Field G

HQ830369

GU723438

99

Fp-H6


HQ830370

GQ121284

99

Fp-H8


HQ830371

GU723438

99

Fp-H9


HQ830372

GQ121284

99

F. proliferatum

cause yellowing symptoms in black pepper (Kueh et al. 1993; Sitepu and Mustika 2000). This study confirmed that F. solani is one of the dominant pathogens in Malaysia black pepper plantations. Fusarium solani is a common soil-borne fungus and a pathogen of many agricultural crops such as pepper (Fletcher 1994). Morphological characteristics, including the presence or absence of microconidia, the shape and size of macroconidia and the morphology, pigmentation and growth rates of the colonies, are used to differentiate Fusarium species (Leslie and Summerell 2006; Nelson et al. 1983).

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Root colonization (%)b

Max identity (%)

In this study, 34 isolates of F. solani were obtained from black pepper plants with yellowing disease symptoms and from rhizosphere soils. Fusarium proliferatum was not isolated from the roots of black pepper plants, and the 19 isolates of F. proliferatum in this study were all isolated from rhizosphere soils. Fusarium solani isolates displayed slight morphological variability with the exception of isolate Fs-H6. Colonies of Fs-H6 were dense and floccose and produced no pigmentation. Isolates of F. proliferatum had variable pigmentation; however, only minor morphological differences were observed.


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Fp-D2 Fp- GU723438 Fp-F3 Fp-G3 Fp-B1 100

Fp-F5

F. proliferatum

Fp-G2 Fp-H6 Fp-A1 Fp-H9 Fp-H8 Fs-H6 Fs-H5 96 100

Fs- FJ719812 Fs-H8 Fs-F5

49

Fs-H4 84

F. solani

Fs-K6 Fs-B3

88

Fs-K2 Fs-E2 Fs-H7

Fusarium oxysporum HQ108020

0.01

Substitutions per site

Fig. 3 Phylogenetic tree based on the ITS and 5.8S rDNA sequences. Number at each branch node indicates the confidence values (%) from bootstrap analysis using 1,000 replications. Outgroup: Fusarium oxysporum

Black pepper plants were susceptible to F. solani, displaying yellowing symptoms and severe rotting of the roots 4 months after inoculation. However, only 8.3 % of the inoculated plants became flaccid and died. Reisolation of the fungus from infected roots on PCNB medium produced colonies of F. solani, thus confirming the identity of the causal pathogen and satisfying Koch’s postulates. Black pepper seedling infection by F. solani significantly reduced the root mass of black pepper. Decreased root mass has also been reported in shisham (Dalbergia sissoo Roxb.) plants inoculated with F. solani (Rajput et al. 2008). In contrast, Kueh and Ahmed (1985) reported that F. solani and F. moniliforme isolated from the roots of infected black pepper vines did not prove to be pathogenic when tested on 3-month-old potted pepper (cv. Kuching) cuttings. The failure of these organisms to infect the plants could be attributed to the unfavourable environment in the glasshouse and a high soil temperature in the pots (Kueh and Ahmed 1985).

Isolates of F. solani that produced red to wine pigmentation in culture were more virulent than other isolates. Duarte and Archer (2003) reported that black pepper cuttings inoculated with three types of F. solani isolates (producing colorless, pink and red pigmentation) varied in the severity of symptoms, leading to the supposition that coloured toxic metabolites might be associated with virulence. According to Duarate and Archer (2003), different isolates of the pathogen also produced different quantities of toxic metabolites, and isolates producing colorless or pink pigmentation were less virulent. PCR-based detection techniques have provided an alternative to morphological identification of Fusarium species (Abd-Elsalam et al. 2003). The low rate of polymorphism in the rDNA transcription unit makes rDNA useful for interspecific comparisons and allows characterization of the rDNA of each species using only a few specimens. As a result, rDNA can provide information about any systematic level (Hillis and Dixon 1991). The internal transcribed spacer region of rDNA is the most used

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target sequence in the molecular detection of fungi and is also the most employed marker used to infer lower-level taxonomy in fungi (Bruns 2001). The ITS and 5.8S regions of rDNA are useful for Fusarium species identification and are used in population genetic analyses of phylogenetic relationships on the species level and below (O’Donnell and Cigelnik 1997; O’Donnell et al. 2000; White et al. 1990). Using the ITS1 and ITS4 primers, we obtained products that were ca. 550–570 bp long for all isolates of Fusarium tested. Lee et al. (2000) reported that after amplification with ITS1 and ITS4 primers, a product of ca. 550 or 570 bp was obtained from F. solani and F. proliferatum isolates. The uniformity of ITS fragment size across several fungal groups makes nucleotide sequencing of ITS fragments necessary to reveal interspecific and, in some cases, intraspecific variation (Batista et al. 2008; Henry et al. 2000; Hinrikson et al. 2005b; Inglis and Tigano 2006; Radford et al. 1998). In the present study, the sequenced ITS and 5.8S rDNA regions exhibited a high level of identity for Fusarium species. Multiple sequence alignments of F. solani and F. proliferatum species using Clustal W (version 1.8) demonstrated that the sequence variation between these two species is due to deletions and insertions in the ITS1 and ITS2 regions. Robin et al. (2000) reported that mutations occurring in the ITS sequence are base insertion or deletion events. The sequence variations of ITS and 5.8S rDNA regions between the F. solani and F. proliferatum isolates were high and thus can be used to build a phylogenetic tree. Isolate Fs-H6, which displayed high morphological differences compared to other F. solani isolates, appeared more distant. Therefore, the ITS1, 5.8S rDNA, and ITS2 regions of ribosomal DNA are useful for identifying the F. solani and F. proliferatum isolates. This result suggests that other species of Fusarium are needed for an exact determination of which regions of rDNA (ITS1 or ITS2) are more variable and informative for phylogenetic analysis. The greatest degree of differentiation among species was obtained using combined ITS1 and ITS2 sequence data, because the sequence length of any rDNA region alone could not be reliably used to discriminate among all species examined (Hinrikson et al. 2005a). A search of similar ITS sequences using the BLAST program confirmed the morphological identification of F. solani and F. proliferatum isolates from black pepper plants. Our results indicated that sequence similarity was not related to the geographical origin of the isolates studied. ITS region sequence analysis can be used to differentiate Fusarium at the species level, and the nucleotide sequence analysis of rDNA regions is useful for determining phylogenetic relationships.

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