Genus Curvularia and Pestalotiopsis as the primary pathogen of leaf spot disease on oil palm seedlings throughout Indonesia

Leaf spot is a major plant disease that affects oil palm seedlings. Several fungal genera, including Cercospora, Curvularia, Drechslera, Pestalotiopsis, and Phyllosticta, have been linked to leaf spot disease in oil palms worldwide. A survey was undertaken in several oil palm nurseries throughout Indonesia to discover the primary pathogen of the leaf spot disease. The result revealed that two dominating genera, Curvularia and Pestalotiopsis, were responsible for leaf spot infections on oil palm seedlings in Indonesia. Curvularia was isolated from oil palm nurseries in Sumatra, Java, Kalimantan, and Sulawesi. Curvularia isolates can be divided into five morphotypes based on colony growth and appearance. On the other hand, three major morphotypes of Pestalotiopsis were collected from Sumatra, Java, Kalimantan, Sulawesi, and Papua.


Introduction
Leaf spot is a frequent oil palm disease in the nursery.The disease poses a serious threat to oil palm seedlings if left untreated.In most cases, seedling mortality is common when leaf spots emerge in the pre-nursery (PN) stage up to the first three months in the main nursery (MN) [1].The probability of plant death increases when the leaf spot coexists with the anthracnose disease [2].At severe disease intensity, the affected seedlings will lose many leaves and thereby require a longer time to recover before being transplanted to the field.
Several genera of pathogenic fungi have been identified as the causative agent of leaf spot disease on oil palms worldwide.These include the genus Cercospora, Curvularia, Drecshlera, Neopestalotiopsis, Nigrospora, Oxydothis, Pestalotiopsis, Phoma, and Phyllosticta [3][4][5][6][7][8][9][10]. In Indonesia, genus Curvularia is frequently reported as the primary pathogen of leaf spot disease in oil palm nurseries in Sumatra and Kalimantan [2].Meanwhile, there is a lack of information on the pathogen of leaf spot disease in the oil palm nursery on other islands such as Java, Sulawesi, Maluku, and Papua.However, without a systematic identification process, many planters believe that the genus Curvularia is responsible for the prevalence of leaf spot disease in oil palm nurseries.To minimize misinformation, more study into the causative agent of leaf spot disease in oil palm nurseries across Indonesia is needed.

Sample collection
Field surveys were conducted in oil palm plantations in Sumatra (Aceh, North Sumatra, Riau, South Sumatra, and Lampung Province), Java (West Java Province), Kalimantan (West, Central, and North Kalimantan Province), Sulawesi (South Sulawesi Province), Maluku and Papua Island (South Papua Province) between September 2022 to February 2023.Leaf samples were collected from oil palm seedlings showing leaf spot symptoms.Symptomatic leaves were preserved in a cooler box before being transported to the laboratory for further examination.

Isolation of the pathogens
Leaf samples from each location were grouped based on the similarity of the symptom appearance.Leaf tissue was cut at approximately 5x5 mm in the region between the diseased and the healthy tissue.Tissue samples were soaked for 30 seconds in NaClO 1%, then in ethyl alcohol 70% for 30 seconds before being rinsed twice in sterile water.The tissues were subsequently cultured on potato dextrose agar (PDA) and incubated for three days at room temperature.A series of subculture were then conducted until the pure culture of suspected pathogens were obtained.Each fungal culture was subjected to Koch Postulate on oil palm leaf to determine the association with the leaf spot disease.Morphological characterization was then conducted based on the colony and spore appearance.

Results and discussion
A total of 205 fungal cultures were isolated from oil palm seedlings in Sumatra, Java, Kalimantan, Sulawesi, Maluku, and Papua.Of these isolates, the genus Curvularia and Pestalotiopsis were the two dominant pathogens associated with the leaf spot of oil palm seedlings in Indonesia, accounting for 47,32% and 41,95%, respectively (Table 1).Curvularia leaf spots mainly occurred in the western part of Indonesia, particularly in Sumatra and Kalimantan Island (Figure 1).On the other hand, Pestalotiopsis leaf spot has a broader distribution but is found to be dominant in the eastern part of Indonesia, from Sulawesi to Papua.Curvularia leaf spot is characterized by a blackish to dark brown necrotic spot with a yellow margin (Figure 2. A-E).The central part of the spot mostly has a lighter color and sometimes develops into a whitish spot (Figure 2.A/B.1).At severe intensity, the spots may coalesce, causing the leaf blades to dry out (Figure 2.B.1).In this study, Curvularia isolates were grouped into five different morphotypes (M1-M5) based on the colony or conidia characteristics.The colony of all morphotypes has a circular form, except M3, which showed an irregular shape (Figure 2.C.2).Colony M1 and M4 have a light to dark brown color with a flat-thin layer of mycelia on the agar surface.In contrast, colonies M2 and M3 have a dark-black color and denser mycelia.The colony M5, on the other hand, was soft-black with curled margin.The conidiophore was straight or flexuous, simple or often geniculate, septate, and pale to brownish in color.Conidia consist of 3-5 cells, ovoid to ellipsoidal, curved, rounded at the end, and pale to dark brown.Conidia are produced singly or in groups on the end of the conidiophore.Mass of conidiophore can be produced on both sides of the leaf surface but is tend to be denser on the adaxial side.Meanwhile, the Pestalotiopsis leaf spot is characterized by circular to elliptical dark brown or black spots with yellow margins (Figure 3).The central part often becomes whitish as the spot expands.Several spots may coalesce at severe intensity, forming a blight symptom.Conidiomata or acervuli were found on the abaxial and adaxial sides of the leaflet.From the observation, Pestalotiopsis cultures were 1308 (2024) 012018 IOP Publishing doi:10.1088/1755-1315/1308/1/0120184 grouped into three different morphotypes (P1-P3).On PDA, the colony of Pestalotiopsis was grown in circular form with an even or undulating margin.Acervuli were produced on the aerial mycelia, containing black conidial masses (Figure 3.A-C.2).Meanwhile, on the leaf surface, acervuli were formed on the necrotic tissue, either on the abaxial or the adaxial side.Conidia were straight, sometimes slightly curved, and contained 5-6 cells.The middle segment (3-4 cells) was pale brown, while the basal and apical cells were hyaline.There were 2-3 tubular appendages on the apical side of the conidium.The genus Curvularia and Pestalotiopsis were previously identified as the pathogen of leaf spot disease on oil palm in Southeast Asia (Indonesia, Malaysia, and Thailand), South America (Columbia, Honduras, Peru, and Venezuela), and Africa (Ghana) [5,7,9,[11][12][13][14][15][16][17].Several species of Curvularia are known to cause severe damage to oil palm seedlings, for instance, C. oryzae, C. eragostidis, and C. lunata [5,15,18].The latter is also responsible for high yield losses on maize, particularly in China [19].On the other hand, several species of Pestalotiopsis, including P. microspora, P. theae, and P. palmarum, were reported to cause severe damage to oil palm seedlings [7,12,14,20].In the field, Pestalotiopsis can be dispersed by insect vectors, such as Leptopharsa gibbicarina (Hemiptera: Tingidae) [21] and several Lepidopteran insects [16].

Conclusion
This study is the first to report the distribution of leaf spot disease in oil palm nurseries in Indonesia.Curvularia and Pestalotiopsis are the two prominent genera associated with leaf spot disease on oil palm seedlings in Indonesia.More research is needed to discover the fungus species, its characterization and pathogenicity as well as its mitigation in the field.

3 Figure 1 .
Figure 1.Distribution map of Curvularia and Pestalotiopsis leaf spot on oil palm seedlings in Indonesia.

Figure 2 .
Figure 2. Five morphotypes of genus Curvularia associated with leaf spot disease on oil palm seedlings in Indonesia.Letters A-E refer to Morphotype 1-5, respectively; (1) Leaf spot symptom on seedling leaflet; (2) Colony growth on PDA at 14 days after incubation at room temperature; (3) Conidia of each morphotype under a microscope.

Figure 3 .
Figure 3. Three common morphotypes of the genus Pestalotiopsis are associated with leaf spot disease on oil palm seedlings in Indonesia.Letters A-C refer to Morphotype 1-3, respectively; (1) Leaf spot symptom on seedling leaflet; (2) Colony growth on PDA at 28 days after incubation at room temperature; (3) Conidia of each morphotype under a microscope.

Table 1 .
Two dominant fungi associated with leaf spot disease on oil palm seedlings in Indonesia