Common Spear Rot of Oil Palm in Indonesia - APS Journals

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Oct 31, 2011 - palm (Elaeis guineensis Jacq.) throughout the world (3,5,6). Since the first report of this disease in Indonesia in the 1920s (11), the disease has ...
Common Spear Rot of Oil Palm in Indonesia Suwandi, Laboratory of Plant Pathology, Faculty of Agriculture, Sriwijaya University, Jl. Palembang-Prabumulih Km.32, Indralaya, Palembang 30662, Indonesia; and Seishi Akino and Norio Kondo, Laboratory of Plant Pathology, Division of Bioresources and Product Science, Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan

Abstract Suwandi, Akino, S., and Kondo, N. 2012. Common spear rot of oil palm in Indonesia. Plant Dis. 96:537-543. Common spear rot (CSR), which is also known as crown disease, was first reported in Indonesia in the 1920s. It has caused considerable losses in young oil palm plantings, and yet the pathogenic agent has remained elusive. Symptomatic spear leaves were collected from oil palm plantations and farm plots in South Sumatra, North Sumatra, and Bangka-Belitung, Indonesia. Of the 14 different fungi isolated, Fusarium incarnatum, F. solani, an undescribed Fusarium sp., and Ceratocystis paradoxa were isolated most frequently from diseased leaf tissue. F. incarnatum and the undescribed Fusarium sp. were also frequently isolated from healthy leaf tissue, along with Pestalotiopsis microspora and Curvularia affinis. Ceratocystis paradoxa was never

isolated from healthy leaf tissue. Koch’s postulate experiments showed that C. paradoxa was able to infect wounded oil palm leaves causing a symptom of extensive rotting similar to that found in the field. Although isolated less frequently and less virulent than C. paradoxa, F. sacchari was also capable of causing lesions on succulent wounded, inoculated leaves. For both C. paradoxa and F. sacchari, the disease severity index was greater when the oil palm leaves appeared to have more succulent growth. Likewise, other Fusarium species and other nonfusarial fungi that were usually not pathogenic were weakly virulent on palms with more succulent growth. These findings confirm that C. paradoxa is one pathogen that is associated with CSR of oil palm in Indonesia.

Common spear rot (CSR), which is also known as crown disease, is the most frequently observed disease affecting young oil palm (Elaeis guineensis Jacq.) throughout the world (3,5,6). Since the first report of this disease in Indonesia in the 1920s (11), the disease has spread throughout the country, especially in Sumatra. The disease has attracted considerable attention because recent commercial oil palm cultivars in Indonesia have been improved from a susceptible mother plant, Deli dura (6). Symptoms of CSR include the development of necrotic lesions on spear leaf leaflets, followed by extensive rotting of leaflets as the leaf expands. This results in a leaf where many of the leaflets have been destroyed, leaving partial or no leaflet tissue attached to the rachis. Under favorable conditions for disease, the rachis develops a characteristic bending in its central portion and, in severe cases, all of the leaves surrounding the spear may be bent down (6,14,21,22). These symptoms result in a unique appearance of the palm crown, and consequently, the disease has been referred to as “crown disease” (22). To distinguish these symptoms from other spear rots and bud rots, Chinchilla (4) proposed using “common spear rot” to describe the spear rot disease with or without rachis bending where the rotting does not progress toward the main meristem. Common spear rot includes crown disease and wither tip, a variation of CSR where tips of the youngest leaves also rot. The disease may infect nursery seedlings, and the peak of incidence in the field occurs between 12 and 18 months after transplanting from the nursery (3,14,19). This disease rarely kills the oil palm, and ultimately, successive leaves are produced without symptoms, but it does significantly reduce growth and yield. Breure and Soebagjo (3) noted that individual palms affected by the disease in two commercial oil palm plantings in North Sumatra yielded 18% fewer cumulative bunches for the first 6 years. In Costa Rica, Chinchilla et al. (5) reported that spear rot with or without rachis bending reduced the cumulative yield 20% during

the first 38 months, with individual palms affected both in the nursery and in the field. The role of agronomic and genetic predisposition factors for the disease have been well studied (1–3,21), but very little research has examined the pathogenic agents. No pathogen has yet been identified as the primary cause of the disease. Fusarium oxysporum and F. solani (21), and Fusarium spp. and Erwinia spp. (14) were the most frequently recovered microorganisms from diseased leaves of oil palm affected by CSR, but the symptoms could not be consistently reproduced by artificial inoculation. No attempt has been made to correlate fungal flora from diseased and healthy tissue of oil palm leaves in previous studies. In this study, fungal species isolated from necrotic lesions on the leaflets and rachises affected by CSR, as well as fungi isolated from asymptomatic oil palm leaves, were identified and the pathogenicity of the isolates was determined.

Corresponding author: Suwandi, E-mail: [email protected] Accepted for publication 31 October 2011.

http://dx.doi.org/10.1094 / PDIS-08-10-0569 © 2012 The American Phytopathological Society

Materials and Methods Collection of leaves and fungal isolation. Symptomatic spear leaves were collected from oil palm plantations and farm plots in South Sumatra (five plots and two plantations), North Sumatra (one plantation), and Bangka-Belitung Province (two plantations), Indonesia, in 2007 and 2008. The palms were 10 months old (nursery seedling) to 8 years old (mature palm). Three to 15 diseased palms were sampled for each plantation and plot, resulting in 114 sampled palms. Asymptomatic spear leaves were collected from the same fields of two oil palm plots in South Sumatra. Ten and 11 healthy palms were sampled from each plot. Portions of diseased spear leaf (both leaflet and rachis tissue) or the asymptomatic spear leaf were cut from each palm, sealed in a plastic bag, and stored in a cooler box. Isolations were made within 24 h of cutting. From each sample, four to five sections (approximately 5 by 5 mm) were cut from the margins of lesions or from the margin of the asymptomatic leaflet lamina. The leaf sections were surface-sterilized with 1% NaOCl for 2 min, rinsed once with sterile distilled water, and blotted dry on a sterile filter paper. The leaf sections were placed on 2% water agar (WA) or 2% malt extract agar (MEA) containing 0.1% streptomycin-sulfate and 0.012% neomycin-sulfate. No specific attempt was made to isolate oomycetes. The agar plates were incubated for 7 days at 27°C in the dark and examined daily for fungal growth. Each distinct fungal colony was subcultured on potato dextrose agar (PDA) and incubated for another 7 to 14 days. Plant Disease / April 2012

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Single spore or hyphal tip subcultures were maintained for routine use on synthetic nutrient agar (SNA) (15) slants in the case of Fusarium spp. and on MEA slants for other fungal genera. Initial identification and cultural characteristics. In all, 114 and 21 spear leaves representing the diseased and healthy palms, respectively, were used to estimate the frequency of occurrence of fungi, with each palm considered a sample unit. Isolated fungi were initially identified based on morphological and cultural characteristics. Fusarium spp. were identified from cultures grown on carnation leaf agar (CLA), SNA, and PDA, according to the descriptions of Nelson et al. (15) and Leslie and Summerell (13). Identification of other fungal species was based on isolates cultured on PDA and MEA, according to species descriptions from the monographs of Guba (10), Ellis (8), Sutton (20), and Upadhyay (23). DNA isolation, PCR, and sequence analysis. Representative isolates of the fungi recovered from the diseased and healthy oil palm leaves were then identified based on DNA sequence analysis. Each fungal isolate was cultured in 15 ml of half-strength potato dextrose broth (Difco Laboratories, Sparks, MD) in 90-mm plastic petri plates at 25°C for 3 to 5 days. The mycelia were harvested by vacuum filtration and freeze-dried. Total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, except for an additional phenol/chloroform extraction prior to the addition of the Buffer AP3/E. DNA concentration was determined spectrophotometrically. A portion of the translation elongation factor 1α gene (the EF-1α) region of fusarial isolates was amplified by PCR, using primers EF-1 (forward: 5′-ATGGGTAAGGA(A/G)GACAAGAC3′) and EF-2 (reverse: 5′-GGA(A/G)GTACCAGT(G/C)ATC ATGTT-3′) (16). The ITS1/5.8 S rDNA/ITS2 (ITS) region of nonfusarial isolates was amplified by PCR, using ITS1 (forward: 5′-

TCCGTAGGTGAACCTGCGG-3′) and ITS4 (reverse: 5′TCCTCCGCTTATTGATATGC-3′) (24). PCR was performed in 50 μl of reaction mixture containing 10 pmol of both primers, 1.25 U Taq DNA polymerase, 0.2 μM of each dNTP, 2× PCR buffer, and 50 ng of template DNA with a thermal cycler (PC-320, Astec, Fukuoka, Japan). An initial denaturation step for 2 min at 95°C was followed by 30 cycles of denaturation for 20 s at 95°C, annealing for 40 s at 58°C, and extension for 1 min at 65°C, with a final extension step of 5 min at 65°C. Negative controls (no template DNA) were included in every assay. The PCR products, after visualization of aliquots on agarose gels, were purified using the QIAquick PCR purification kit (Qiagen Inc.) according to the manufacturer’s instructions. The EF-1α and ITS regions were sequenced at Hokkaido System Science, Co., Ltd., Sapporo, Japan. Identification of fusarial isolates was accomplished by BLAST searches of the EF-1α sequence on the GenBank database (http://www.ncbi.nlm.nih.gov). Nonfusarial fungi were identified by BLAST searches of the ITS region on the GenBank database. Table 1 lists the GenBank accession numbers for the isolates used in the sequence analyses. Pathogenicity tests. Eighteen representative fungal isolates recovered from diseased leaves and five isolates from the healthy leaves were used to test for pathogenicity of oil palm isolates from Indonesia. Pathogenicity tests were conducted on youngest bifid leaves of seedlings grown in a flooded nursery and spear leaves of seedlings grown in a field nursery. The nurseries contained the oil palm cultivar Dumpy (Dy × P), established in an experimental field at the Faculty of Agriculture, Sriwijaya University, Palembang, Indonesia. Plants in the flooded nursery were grown in 250-ml plastic pots containing local river sand under a 25% shading net. The pots were placed in a tray filled with 1% NPKMgCa fertilizer

Table 1. Frequency of isolation and the GenBank accession number of fungi collected from spear leaves of oil palms in Indonesia Frequency of isolation (%)q Speciesr

Isolates used for DNA sequence analysis

Diseased leaf

Healthy leaf

Isolate

GenBank no.

Fusarium incarnatum

37

38

F. solani

25

5

Fusarium sp.

16

57

F. sacchari F. oxysporum F. proliferatum Ceratocyctis paradoxa

4 4 2 11

0 0 0 0

Lasiodiplodia theobromae Pestalotiopsis microspora

5 8

0 24

Curvularia affinis

5

29

Other identified isolatesz

11

0

BT48 PRT6 LR21 PCO2 S110t FPP1 SB21 B127 SK11 S120u BT38 BT41 FP41 PRT1 S115v SJW1 PRT7 BT40 B116 B125 B118 S143 S145x S255 S160y ...

HM770722s HM770723s HM770724s HM770725s ... HM770726s HM770727s HM770728s HM770729s ... HM770730s HM770731s HM770732s HM770733s ... HM770734s HM770735s HM770736s HM770737w HM770738w HM770739w HM770740w ... HM770741w ... ...

q

Number of palms in which each fungus was isolated/number of palms tested × 100. r Fungal species from oil palm from Indonesia were determined based on morphology and sequence analyses. s Translation elongation factor 1-alpha (EF-1α). t Isolate from the healthy leaf. The sequence had 100% nucleotide identity to isolate BT48. u Isolate from the healthy leaf. The sequence had 100% nucleotide identity to isolate SB21. v Isolate from the healthy leaf. The sequence had 100% nucleotide identity to isolate BT38, BT41, FP41, and PRT1. w Internal transcribed spacer region (ITS1/5.8 S rDNA/ITS2). x Isolate from the healthy leaf. The sequence had 100% nucleotide identity to isolate S143. y Isolate from the healthy leaf. The sequence had 100% nucleotide identity to S255. z Less common isolates, including Rhizoctonia, Nigrospora, Moniliella, and Colletotrichum, which were identified to genera based on morphological characteristics. Those isolates did not produce any rotting symptoms when inoculated on unopened oil palm leaves in preliminary pathogenicity tests. 538

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(16:16:16:1.5:5) and 1% urea, maintained to a depth 2 to 3 cm. The nutrient solution was changed weekly. Plants in the field nursery were grown in nursery bags filled with 10 kg of topsoil. Seedlings were fertilized monthly with 10, 20, and 30 g NPKMgCa fertilizer and 10 g urea per plant for months 1 to 6, 6 to 9, and 9 to 12 after sowing, respectively. The nursery bags were spaced 1 m apart in a triangular pattern. Preliminary tests showed that leaf inoculations with a spore suspension or mycelial plug were ineffective unless the leaf was wounded. Attempts to inoculate Fusarium spp. and C. paradoxa without wounding were conducted on 100 seedlings in the field nursery as well as on 300 cut spear leaflets, but only one seedling inoculated with C. paradoxa showed CSR symptoms. Wound inoculations with Fusarium spp. and C. paradoxa using PDA plugs or injected spore suspensions had similar disease index values as injection inoculations. However, the agar plug inoculation method resulted in larger necrotic lesions on the control palms (inoculated with sterile PDA plugs). Therefore, injection inoculation was used throughout experiments. Leaves were inoculated by injecting ~50 μl spore suspension (106 conidia/ml) using a hypodermic disposable syringe (27 gauge × ½ in.) at the base of an unopened leaf (14). Spores were harvested from 1-week-old PDA cultures of Fusarium and Ceratocystis, and 4-week-old PDA cultures of other genera. For control experiments, leaves were injected with sterile distilled water. Seedlings were inoculated at 3 to 5 or 10 to 12 months of age in the flooded or field nursery, respectively. In the flooded nursery, inoculations were performed on seedlings either without or with

predrainage. For those with predrainage, the nursery was drained 9 days before inoculation and grown without watering for 6 days, and then reflooded with the freshly prepared fertilizer solution. In the field nursery, inoculations were performed either on seedlings in the period of rapid and succulent growth of spear leaves within 7 days after fertilizer application or on seedlings in the period of normal growth of spear leaves at 4 weeks after fertilizer application during the rainy season (November to April). The inoculation experiment was repeated once during the period of normal growth and twice in the period of rapid and succulent growth. Disease severity was assessed 12 days postinoculation on a scale of 1 to 5, where 1 = no lesion or only a very small lesion under 5 mm in length, 2 = a restricted lesion 5 to 15 mm in length, 3 = a medium lesion 15 to 30 mm in length, 4 = a large lesion greater than 30 mm or extensive rot on fewer than 50% of the leaflets, and 5 = a large lesion or extensive rot on more than 50% of the leaflets. Sections were cut from the margins of 10 representative lesions per isolate, surface-sterilized, and plated on Nash-Snyder medium (15) in an attempt to recover the inoculated Fusarium or plated on MEA to recover nonfusarial species to complete Koch’s postulates. Fungal identity was verified by colony and conidial morphology. Fusarium species are frequently isolated from healthy oil palm leaves, and thus naturally colonizing strains could interfere with the recovery of the inoculated strain. Therefore, nitrate-nonutilizing (nit) mutants were used, in addition to the wild types, to verify the colonization of inoculated strains to fulfill Koch’s postulates for Fusarium isolates. Nit mutants were derived from wildtype culture by growing on minimal medium (MM) with chlorate,

Fig. 1. Symptoms of common spear rot (CSR) of oil palm. A, Rotted and disintegrated leaflets of emerging spear leaf of a 4-year-old tree in the field. B, A bent rachis with the stumps of pinnae, and rotted and disintegrated distal parts of fronds of a 15-month-old seedling in the field nursery. C, Restricted lesion (left) and extensive rot (right) on youngest bifid leaf 6 days after inoculation. D, Medium lesion. E, Extensive rot on spear leaf 12 days after inoculation. Plant Disease / April 2012

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as described by Correll et al. (7). Only stable nit mutant cultures that showed similar growth rate to the wild type were used. The nit mutants were inoculated on 22 and 31 field nursery seedlings in the period of rapid and succulent growth. Inoculation, disease scoring, and reisolation were done following the same procedure as described above for the wild types. Phenotypes of the nit mutants recovered from the lesions were identified based on their sparse growth on MM (7,13). To verify contamination by other nit mutants or reinfestation with natural Fusarium species, all recovered nit mutants and wild types were grown on PDA and SNA to compare their morphology with the original wild types. This experiment was repeated once. Pathogenicity of isolates was tested also in mixed inoculation assay with 13 combinations. Spore suspensions (106 conidia/ml) from tested isolates were mixed in equal volume and then used for injection inoculation as described above. Each isolate mixture was inoculated on 15 and 42 field nursery seedlings in the period of rapid and succulent growth. This experiment was repeated once. One-way analyses of variance (ANOVA) were performed in order to assess differences in the disease severity index from each trial. A two-sample t test with unequal variance was used to analyze the difference of disease severity index either between the nursery without predrainage and with predrainage or between the nursery in a period of normal growth and a period of rapid growth. The Tukey’s test was used for comparison of the disease severity index in mixed-inoculation test. Analyses were implemented using R statistical software (version 2.11.1; R Foundation for Statistical Computing, Vienna).

Results Field observations and symptom development. Diseased oil palms showing typical CSR symptoms were observed in oil palm plantations and farmer plots located in three regions in Indonesia: South Sumatra, North Sumatra, and Bangka-Belitung Province. Disease incidence reached 10% or more in the fields of South Sumatra. The disease incidences in the other locations were less than 5%. The disease was found affecting all surveyed young plantings (under 4 years old), including the seedlings in the main nursery. In the fields of South Sumatra, the disease was found even on mature plants (8 years old). Common spear rot symptoms were characterized by extensive rotting of spear leaflets, followed by shriveling and collapse of the leaf tissue. This resulted in a leaf where many of the leaflets were destroyed, leaving just partial or no leaflet tissue attached to the rachis (Fig. 1A). Under favorable conditions, severe rotting of the leaflets usually coincided with the onset of an abnormal bending of the young rachis (Fig. 1B). The symptom of rachis bending was found in 25% (29 out of 114) of sampled CSR palms. Isolation, morphology, and identification of the causal fungi. Ninety-eight fungal isolates were obtained from symptomatic spear leaves. The identification by sequence analysis corresponded well with morphological identification. Nine genera and 15 species were identified based on morphological characteristics and BLAST searches on the GenBank database (Tables 1 to 3). Fusarium was the dominant fungal genus isolated from CSR palms, although no attempts were made to isolate oomycetes. Members of the genus

Table 2. Characteristics and identification of Fusarium species from spear leaves of oil palm in Indonesia Character Macroconidia Septum Size (μm)y Shape Apical Basal Microconidia Septum Size (μm)y

F. incarnatumx

F. solanix

Fusarium sp.

F. saccharix

F. oxysporumx

F. proliferatumx

3–5, mostly 3 3 septa (31.5 ± 3.3 × 3.7 ± 0.3) Straight to slightly curved Curved Foot shaped or notched

3 3 septa (26.2 ± 2.1 × 5.5 ± 0.3) Stout, thick walled

3–5, mostly 3 3 septa (38.1 ± 4.8 × 3.7 ± 0.3) Slightly falcate

3 3 septa (35.4 ± 4.6 × 3.7 ± 0.5) Slightly falcate

Blunt and rounded Notched

Hooked Foot shaped or notched

Curved Poorly developed

3 3 septa (33.5 ± 5.5 × 4.0 ± 0.4) Straight to slightly curved Tapered and curved Foot shaped

3 3 septa (39.5 ± 7.7 × 3.9 ± 0.3) Straight to slightly curved Curved Notched

... ...

0–1, mostly 0 0 septum (9.2 ± 1.8 × 2.8 ± 0.4) Oval-allantoid

0–2, mostly 0 0 septum (8.2 ± 1.6 × 2.2 ± 0.3) Oval

0–1, mostly 0 0 septum (8.3 ± 1.9 × 2.6 ± 0.3) Oval-kidney shaped Short monophialide

0–1, mostly 0 0 septum (8.6 ± 2.2 × 2.5 ± 0.3) Club shaped with a flattened base Mono- and polyphialide

Shape

...

0–2, mostly 0–1 1 septum (10.5 ± 1.8 × 3.9 ± 0.5) Oval-ellipsoid

Phiallide

...

Long monophialide

Mono- and polyphialide

Mono- and polyphialide

... ...

... ...

... ...

...

...

...

...

Phiallide

Polyphialide

...

...

...

...

Chlamydosporey

...

...

67 ± 3 Deep olive-buff to tan F. incarnatum (13; 92–99; 953–1219; 0.0)

49 ± 3 Orange to vinaceous purple Fusarium sp. ECYL-2007d (4; 96; 1020–1057; 0.0)

58 ± 2 Pale violet

Mostly terminal, singly or in pairs until 5, diam. 7.5 ± 0.5 µm 52 ± 1 Pale violet

...

Colony diameterz Colony color (top view) BLASTN search ID (no. of hits of the top 100 matches; % similarity; bit score; E-value)

Mostly terminal, singly or in pairs until 3, diam. 7.4 ± 0.7 µm 41 ± 2 Cream

0–1, mostly 1 1 septum (17.2 ± 3.8 × 4.2 ± 0.5) Curved or comma shaped Mono- and polyphialide ...

... ...

Shape

1–2 1 septum (18.1 ± 3.4 × 3.0 ± 0.3) Fusoid

50 ± 2 Yellowish orange

F. sacchari (19; 98–99; 1134–1206; 0.0)

F. oxysporum (20; 99; 1214–1236; 0.0)

F. proliferatum (39; 98–99; 1075–1229; 0.0)

Mesoconidia Septum Size (μm)y

x y z

F. solani (37–80; 97–100; 1171–1255; 0.0)

The morphological characteristics match the species description from the monographs of P. E. Nelson et al. (15), and J. F. Leslie and B. A. Summerell (13). Mean and standard deviation (SD) of 50–100 spores. Mean and SD of five PDA cultures at 25°C in the dark for 4 days.

540

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Fusarium were isolated from 80% (91 out of 114) of CSR palms, consisting primarily of Fusarium incarnatum (Desm.) Sacc. (37%), F. solani (Mart.) Sacc. (25%), and an undescribed Fusarium sp. (16%). Of the nonfusarial species, Ceratocystis paradoxa (Dade) Moreau was most frequently recovered from diseased tissue (Table 1). Fusarium species were also the most frequently isolated from asymptomatic spear leaves, dominated by an undescribed Fusarium sp. (57%) and F. incarnatum (38%). F. solani was less frequently recovered from surface-sterilized healthy tissue. For nonfusarial species, only Curvularia affinis and Pestalotiopsis microspora were recovered from the healthy tissue. Isolates from the healthy spear leaves had 100% nucleotide identity to at least one isolate from diseased tissue (Table 1). Pathogenicity tests. In pathogenicity tests, initial symptoms appeared as water-soaked lesions starting from the injected wound within 2 to 3 days after inoculation. Six days after inoculation, 91% of the lesions stopped enlarging and remained small, less than 30 mm in length (Fig. 1C and D). On leaves inoculated with C. paradoxa and F. sacchari, the rotting lesions enlarged rapidly within 6 days after inoculation and turned brownish to black, sometimes covered with whitish spore masses of Fusarium spp. (Fig. 1C and E). This kind of extensive rot was similar to a typical symptom of CSR observed in the field. Once the leaves opened, the lesions stopped growing, except those caused by F. sacchari and C. paradoxa, which continued expanding to develop a yellow patch in the case of F. sacchari and a scorch symptom in the case of C. paradoxa. Both symptoms were observed on CSR palms in the field. The rachis bending associated with severe CSR symptom was not found in 969 inoculated seedlings in the flooded nursery, but three of 657 inoculated palms in the period of rapid and succulent growth in the field nursery exhibited the bent rachis with severe rotting of spear leaf leaflets (length of lesion more than 300 mm within 18 days after inoculation). Two of 74 seedlings in the predrainage flooded nursery inoculated with C. paradoxa died as the rotting extended to the bud area. The control plants, injected with distilled water, remained asymptomatic or had a tiny lesion less than 5 mm in length at the injection site.

Predrainage for 6 days, followed by reflooding with fertilizer solution, promoted the rapid and succulent growth of the bifid leaf. A similar result was observed in the field nursery within 7 days after fertilizer application during the rainy season. Inoculations of some isolates on bifid leaf of the predrainage flooded nursery or most isolates on the actively growing and succulent spear leaves in the field nursery resulted in higher disease severity. A restricted lesion less than 30 mm in length and occasionally the extensive rots of CSR were observed on these susceptible leaves inoculated with tested isolates, while a small lesion less than 10 mm was frequently observed on the normal leaves. Among the tested fungi, only C. paradoxa and F. sacchari were able to induce extensive rot on the normal leaves (Table 4). Based on the disease severity index, and considering that the more virulent isolates are those which were able to produce extensive rot on both bifid and spear leaves of different physiological conditions, C. paradoxa was judged to be the most virulent, followed by F. sacchari as intermediately virulent. Other species are considered as either nonpathogenic or weakly virulent as they failed to induce extensive rot on the normal leaf, and the disease index was less than 2.5 on the susceptible leaf. Similar weak virulence was exhibited by nit mutants of the fusarial isolates (Table 4). Most of the inoculated wild types and nit mutants of Fusarium spp., as well as wild types of the nonfusarial fungi, were consistently reisolated from the lesions (Table 4), even from very small lesions at the injection site. The nit mutants were not reisolated from leaves injected with distilled water or the noninoculated control palms. The wild types of either the same or different species of Fusarium spp. were frequently isolated from lesions from which we failed to recover the inoculated nit-mutants. Inoculation with a mixture of Fusarium isolates or with mixtures of Fusarium and nonfusarial isolates did not increase the disease severity index compared to inoculation with just a single isolate (Table 5).

Discussion Among the fungi isolated from diseased leaves, only C. paradoxa was highly virulent, and only if leaves were wounded prior to inoculation. Each isolate of C. paradoxa consistently caused typi-

Table 3. Characteristics and identification of fungi from spear leaves of oil palm in Indonesia Character

C. paradoxa

Curvularia affinis

L. theobromae

P. microspora

Cylindrical to oval, smoothwalled

Fusiform to curved

Subovoid to ellipsoidoblong, often longitudinal striation

Color

Hyaline to mid-brown

Intermediate cells brown or dark brown, terminal cells subhyaline or very pale brown, the central cell usually darkest and swollen

Hyaline, granulose, cinnamon to fawn when mature

Septum Size (μm)y

0 10.0 ± 2.3 × 5.4 ± 0.7

3–4, mostly 4 20.8 ± 2.4 × 9.7 ± 0.9

0–1, 1 when mature 27.1 ± 1.5 × 14.0 ± 0.7

Fusiform, straight or rarely curved, apical appendages 2– 4, mostly 3, basal appendages straight Olivaceous with the two superior median cells or single median cell darker, or with dark band at septum separating 2 superior median cells; apical and basal cells hyaline 4 23.4 ± 1.8 × 6.3 ± 0.5; apical appendages: 22.3 ± 5.5; basal appendages: 5.9 ± 2.4

Obovate to oval, thickwalled terminal in chain Brown 0 15.1 ± 1.9 × 9.1 ± 0.9 34.5 ± 5.3 Dark olive-gray

...

...

...

... ... ... 6.7 ± 0.2 Buffy olive to orangecinnamon Curvularia affinis Boedijn (8) Curvularia affinis (3; 98–100; 955–974; 0.0)

... ... ... 36.1 ± 1.1 Olive-gray

... ... ... 14.2 ± 0.7 Pale buff

Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (20) L. theobromae (20; 99–100; 918–929; 0.0)

Pestalotiopsis microspora (Speg.) Bat. & Peres (10) P. microspora (23; 99–100; 928–944; 0.0)

Conidia Shape

Chlamydospore Shape Color Septum Size (μm)y Growth rate (mm/day)z Colony color (top view) Morphological identification (References) BLASTN search ID (no. of hits of the top 100 matches; % similarity; bit score; E-value) y z

Ceratocystis paradoxa (Dade) Moreau (23) C. paradoxa (10; 99–100; 710–915; 0.0)

Mean and standard deviation (SD) of 50–100 spores. Mean and SD of five PDA cultures at 25°C in the dark. Plant Disease / April 2012

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cal CSR symptoms, suggesting that this pathogen is a contributing factor to CSR of oil palm in Indonesia. However, this fungus was less frequently recovered from diseased tissue relative to Fusarium spp. C. paradoxa may be less competitive than Fusarium in colonizing oil palm leaf tissue or the Fusarium isolates overgrew C. paradoxa on the medium used for isolation. Sporulation of Fusarium was frequently observed on the lesions where Ceratocystis had been inoculated but not vice versa. C. paradoxa is the most common fungus recovered from oil palm tissues affected by various diseases, but was not known to be associated with lesions associated with CSR. This fungus has been associated with dry basal rot that attacks the stem, leaves, and fruits (18,22) and black rot of sprouted seeds (17), and has been suspected as a possible pathogen of bud rot (fatal yellowing) in Central and South America (9). While F. sacchari showed moderate virulence and was only recovered from one oil palm planting, it has the potential to be widespread, as the nit mutant of this fungus was frequently found on diseased leaves inoculated with other species. Fusarium was the predominant genus isolated from CSR lesions, and F. incarnatum, F. solani, and undescribed Fusarium sp. were the most common species. Fusarium sp. and F. incarnatum also were the predominant species recovered from asymptomatic tissues, suggesting that they are common fungal inhabitants of spear leaf leaflets. Inoculations using both the wild type and nit mutants of Fusarium spp. resulted in lesions that were larger than wound lesions from water controls. As with C. paradoxa and F.

sacchari, lesion size increased significantly when more susceptible leaf tissue was inoculated. Such weakly virulent isolates might be considered weak or secondary pathogens. Failure to induce rotting by inoculation of Fusarium spp. and other fungi isolated from spear rot using the same methods as in this study has been reported (14). The successful inoculation of oil palm leaves with Fusarium sp. and F. oxysporum has been reported in Malaysia to cause symptoms of wither tip (22) and in Congo to cause symptoms of patch yellows (12), in which both diseases have been considered to be CSR variations (22). Because no information was available concerning the condition of the spear leaves that were used in both of these reports, these results could not be compared with our observations. Some of the weak pathogens recovered from CSR lesions are also known to be associated with various other diseases of oil palm, such as Pestalotiopsis species causing leaf spot and gray leaf blight (21), Curvularia species and seedling blight (22), and Lasiodiplodia theobromae of seedling anthracnose (22), although they did not cause the symptoms of CSR. Increased leaf susceptibility was necessary for symptom development by what may be considered to be the less virulent pathogens. Poor drainage and excess nitrogen fertilizer have been reported to be favorable for CSR (1). On the contrary, in our study, poor drainage in the flooded nursery without predrainage did not increase susceptibility. In the flooded nursery, the rapid and succulent growth of oil palm leaves that were susceptible to fungal rotting was accomplished by alternation of flooding and drainage. In

Table 4. Pathogenicity of fungal isolates recovered from oil palms on bifid leaves of 3- to 5-month-old and spear leaves of 10- to 12-month-old seedlings of oil palm Inoculation with wild-type culture DSI on bifid leaf of flooded nursery without or with 6 days predrainagev Fungal species, isolatew F. incarnatum BT48 F. incarnatum PRT6 F. incarnatum LR21 F. incarnatum PCO2r F. incarnatum S110h F. solani FPP1r F. solani SB21 F. solani B127 F. solani SK11 F. solani S120h Fusarium sp. BT41r Fusarium sp. PRT1r Fusarium sp. S115h F. sacchari SJW1 F. oxysporum PRT7r F. proliferatum BT40 Ceratocystis paradoxa B116r C. paradoxa B125 Lasiodiplodia theobromae B118r Pestalotiopsis microspora S143 P. microspora S145h Curvularia affinis S255 Curvularia affinis S260h Distilled water (control) Significancez

Without

With 6 days, Nursery 1

With 6 days, Nursery 2

1.9 (0/19) 1.6 (0/19) 1.9 (0/20) 2.3 (0/14) ... 1.6 (0/20) 2.0 (0/20) 1.3 (0/15) 1.6 (0/19) ... 2.1 (0/19) 2.1 (0/15) ... 2.4 (3/18) 1.8 (0/20) 1.9 (0/15) 3.0 (4/20)

2.2 (3/20) 1.8 (1/17) 2.1 (0/15) 2.1 (4/10) ... 2.3 (3/20)* 2.0 (0/20) 1.9 (0/18)* 1.5 (0/15) ... 2.1 (4/20) 2.2 (1/17) ... 2.7 (5/20) 2.2 (1/20)* 2.1 (1/20) 3.9 (10/17)*

3.0 (6/20) 2.1 (0/15)

Inoculation with nit mutant culture on spear leaf during rapid growth

DSI on spear leaf during normal or rapid growth in field nurseryv Normal

Rapid, Nursery 1

Rapid, Nursery 2

% reisolationx

2.0 (2/30) 1.9 (1/18) 1.9 (0/15) 2.3 (3/20) ... 2.3 (3/20)* 2.0 (0/20) 1.9 (0/18)* 1.3 (0/15) ... 2.3 (3/20) 2.3 (1/18) ... 2.7 (6/20) 1.9 (2/20) 2.1 (0/20) 3.8 (10/18)*

1.3 (0/27) 1.6 (0/28) 1.9 (0/29) 1.8 (0/25) 1.4 (0/15) 1.4 (0/28) 1.2 (0/24) ... 1.3 (0/23) ... 1.7 (0/28) 1.6 (0/25) 1.7 (0/16) 2.1 (1/28) 2.0 (0/23) 1.7 (0/23) 3.0 (2/28)

2.1 (0/19)* 2.2 (2/15)* 2.6 (2/15)* 2.2 (1/20) 1.5 (2/22) 2.2 (1/20)* 2.1 (1/14)* 1.4 (0/16) 1.7 (0/12) 1.3 (0/15) 2.4 (1/20)* 2.1 (0/14)* 1.9 (0/14) 3.2 (8/20)* 2.3 (0/20) 2.1 (0/15)* 3.8 (13/20)*

2.1 (1/20)* 2.1 (2/15) 2.3 (1/15) 2.2 (1/20) ... 2.1 (1/20)* 1.9 (0/15)* 1.6 (0/16) 1.5 (0/13) ... 2.5 (3/20)* 2.2 (0/15)* ... 2.8 (3/20)* 2.1 (0/20) 2.0 (0/15) 3.9 (15/20)*

100 100 100 100 100 100 42 27 100 80 100 100 100 100 100 100 100

2.1 (0/30) 2.4 (4/30) 2.4 (2/29) 1.9 (2/31) ... 2.1 (1/31) 1.2 (0/28) 1.6 (0/22) 1.4 (0/22) ... 2.5 (3/29) 2.4 (3/22) ... 3.0 (6/31) 2.0 (0/30) 1.9 (0/23) ...

4.2 (10/17)* 2.0 (2/20)

3.9 (10/19)* 2.0 (2/20)

2.9 (5/25) ...

3.6 (12/20)* 2.2 (0/19)

4.0 (13/20)* 2.2 (0/20)

100 60

... ...

... ...

1.9 (0/10)

2.1 (0/15)

2.1 (0/15)

1.5 (0/28)

2.4 (0/19)*

2.4 (0/20)*

70

...

...

... 2.3 (0/10) ... 1.0 (0/10) P < 0.001

... 2.1 (2/28) ... 1.0 (0/15) P < 0.001

... 2.2 (2/17) ... 1.0 (0/15) P < 0.001

... 1.1 (0/28) ... 1.0 (0/15) P < 0.001

1.4 (0/23) 2.2 (0/17)* 1.1 (0/16) 1.0 (0/15) P < 0.001

... 2.0 (0/16)* ... 1.0 (0/15) P < 0.001

70 100 50 ... ...

...

...

1.0 (0/15) P < 0.001

... ...

v

DSIv

% reisolationy 100 50 90 85 ... 100 40 70 50 ... 100 65 ... 100 80 100 ...

Disease severity index (DSI) was assessed on inoculated leaves using a scale of 1 to 5, where 1 = no lesion or tiny lesion (less than 5 mm in length), 2 = restricted lesion (length from 5 to 15 mm), 3 = medium lesion (length from 15 to 30 mm), 4 = large lesion or extensive rot (length more than 30 mm) on less than 50% of the leaflets, and 5 = large lesion or extensive rot on more than 50% of the leaflets. Value represent the average disease severity index with number of palms showing extensive rot (disease score 4 and 5) per the total palms inoculated with each isolate in parenthesis. Asterisk indicates that there is a significant difference (P < 0.05) on disease severity index either between the nursery without predrainage and with predrainage or between the nursery in period of normal growth and in period of rapid growth according to a two-sample t test with unequal variance. w Source of isolation, r = isolate from diseased rachises, h = isolate from the healthy leaves, and the rests from diseased leaves. x Percentage of wild-type culture recovered from 10 lesions of inoculated spear leaves in each experiment. y Percentage of nit mutant cultures recovered from 20 lesions of inoculated spear leaves. z One-way analyses of variance (ANOVA) were used to examine the severity index in each experiment. 542

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the field nursery, the susceptible leaves were produced during the period of rapid growth after fertilizer application. These results confirm the previous report that the peaks of CSR incidence in the fields coincided with the onset of the rainy season and increased fertilizer applications (1). Isolates of C. paradoxa from spear rot either with (B116) or without (B125) rachis bending caused the rotting symptom on both bifid and spear leaves, but did not induce rachis bending. In the field surveys, spear rot without rachis bending was frequently observed, although the disease was more severe on the palms with rachis bending. Rachis bending with severe rotting of the leaflets was observed in three palms inoculated with Fusarium sp. and F. incarnatum. The genetic (progenies of Deli dura) and environmental factors (onset of the rainy season and the fertilizer applications) have been known to predispose spear leaves to both spear rot and the rachis bending (1,5,19), suggesting a close association between the two. However, these findings are inadequate to establish a correlation between rachis bending and fungal rotting, which needs further study. Although spear rot with rachis bending causes more economic loss than spear rot alone (19), the use of the name CSR is appropriate to describe the diseases until the association between rachis bending and fungal rotting can be proven.

Table 5. Single and mixed inoculation of fungi from oil palm on spear leaves of oil palm seedlings Inoculation Trial 1, inoculations of Ceratocystis paradoxa with Fusarium spp. C. paradoxa B125 C. paradoxa B125 + Fusarium sp. BT41 + F. solani FPP1 C. paradoxa B125 + Fusarium sp. BT41 + F. oxysporum PRT7 Trial 2, inoculations of Fusarium sacchari with other Fusarium spp. F. sacchari SJW1 F. sacchari SJW1 + Fusarium sp. BT41 + F. solani FPP1 F. sacchari SJW1 + Fusarium sp. BT41 + F. oxysporum PRT7 Trial 3, inoculations of Lasiodiplodia theobromae with Fusarium spp. L. theobromae B118 L. theobromae B118 + Fusarium sp. BT41 + F. solani FPP1 L. theobromae B118 + Fusarium sp. BT41 + F. oxysporum PRT7 Trial 4, inoculations of Pestalotiopsis microspora with Fusarium spp. P. microspora S143 P. microspora S143 + Fusarium sp. BT41 + F. solani FPP1 P. microspora S143 + Fusarium sp. BT41 + F. oxysporum PRT7 Trial 5, inoculations between Fusarium spp. F. incarnatum BT48 Fusarium sp. BT41 F. solani FPP1 F. oxysporum PRT7 F. incarnatum BT48 + Fusarium sp. BT41 F. incarnatum BT48 + F. solani FPP1 F. incarnatum BT48 + F. oxysporum PRT7 Fusarium sp. BT41 + F. solani FPP1 Fusarium sp. BT41 + F. oxysporum PRT7 z

Disease severityz

3.9 (12/15) a 3.1 (8/31) b 2.6 (3/32) b

2.7 (3/19) a 2.3 (2/39) ab 2.0 (0/38) b

2.4 (2/20) a 2.2 (0/40) a 2.1 (0/39) a

2.5 (1/18) a 2.1 (1/40) a 2.3 (2/40) a 2.2 (0/34) b 2.5 (8/40) a 2.1 (0/30) b 2.1 (1/29) b 2.1 (2/35) b 2.4 (3/42) ab 2.3 (2/34) ab 2.3 (2/30) ab 2.2 (0/33) b

Disease severity index was assessed on inoculated spear leaves of field nursery in period of rapid growth using a scale of 1 to 5, where 1 = no lesion or tiny lesion (less than 5 mm in length), 2 = restricted lesion (5 to 15 mm), 3 = medium lesion (l 15 to 30 mm), 4 = large lesion or extensive rot (more than 30 mm) on less than 50% of leaflets, and 5 = large lesion or extensive rot on more than 50% of leaflets. Values represent average disease severity index with number of palms showing extensive rot (disease score 4 and 5) per total palms inoculated in parentheses. Disease severity index followed by different letter in columns by trial are significantly different (P < 0.05) according to Tukey’s test.

The present study confirms that C. paradoxa can cause CSR when the leaf tissue is wounded prior to inoculation. Some fungi that were recovered from both diseased and healthy tissue, such as F. incarnatum and an undescribed Fusarium sp., may be weak pathogens when leaf tissue is succulent and wounded. Mixed inoculation using multiple Fusarium isolates or between Fusarium and nonfusarial isolates, including C. paradoxa, did not increase disease severity, suggesting that coinfection of multiple pathogens may be of minor importance in pathogenesis of CSR. Further investigations are needed to assess the interactions between predisposing factors and the pathogenic potential of weak pathogens. Effect of water-related stress predisposition on the virulence of C. paradoxa and weak pathogens is currently under investigation.

Acknowledgments We thank the editors and reviewers for critical review of the manuscript. Suwandi received a doctoral fellowship (no. 1668.4/D4.4/2008) from the Indonesian Directorate General of Higher Education (DIKTI).

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