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Identification of Pythium and Phytophthora Associated with Durian (Durio sp.) in Indonesia: Their Molecular and Morphological Characteristics and Distribution



Panca J. Santoso, I. Nyoman P. Aryantha, Adi Pancoro and Sony Suhandono
 
ABSTRACT
Phytophthora and Pythium are reported as pathogen causing tree-decline to durian. Survey relating to their diversity and distribution in Indonesia is very limited. A research to identify their molecular, morphological characteristic and distribution was undertaken from 2011 to 2014. Molecular identification was based on ITS-nrDNA sequences. Morphological characteristics observed were colony motif, shape and sporangium size. A total of 36 isolates were successfully baited from 32 durian fields represent of 17 provinces in Indonesia. Based on ITS-nrDNA sequences, the isolates correspond to six Pythiaceae species, namely Pythium cucurbitacearum, Pythium vexans, Pythium sp. D37, Pythium deliense, Phytophthora cinnamomi var., parvispora and Phytophthora palmivora. These species demonstrated the diversity of Pythiaceae associated with durian in Indonesia. The diversity was also confirmed by the morphological characteristics such as colony motif, shape and sporangium size. Pythium cucurbitacearum were found in 13 (76.5%) provinces and Pythium vexans were in 10 (58.8%). The findings concerning the distribution of both pathogens are indicating that these two species could be more dangerous than Phytophthora palmivora. This is the first time; Pythium cucurbitacearum, Pythium sp., D37, Pythium deliense, Phytophthora cinnamomi var., parvispora reported their association with durian.
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Panca J. Santoso, I. Nyoman P. Aryantha, Adi Pancoro and Sony Suhandono, 2015. Identification of Pythium and Phytophthora Associated with Durian (Durio sp.) in Indonesia: Their Molecular and Morphological Characteristics and Distribution. Asian Journal of Plant Pathology, 9: 59-71.

DOI: 10.3923/ajppaj.2015.59.71

URL: http://scialert.net/abstract/?doi=ajppaj.2015.59.71
 
Received: February 23, 2015; Accepted: April 28, 2015; Published: June 11, 2015

INTRODUCTION

Durian (Durio sp.) is a tropical fruit crop which is highly prized culturally and economically in South East Asia. The fruit is very famous not only due to the taste richness but also the strong odour. Durian is believed native to Kalimantan or Borneo Island (Kostermans, 1958; Brown, 1997) and standard Indonesia as the third largest durian producer in the world after Thailand and Malaysia (Somsri, 2014). Total area planted durian in this country reached about 69.045 ha which produce fruit a total of 883.969 t in year 2011 (MoA., 2012).

Durian culture practice in Indonesia is now moving from traditionally toward more intensively. This practice is delivering hope to increase the fruit quantity and quality. On the other hand, intensive practices such as monoculture planting and high fertilizer applications could lead the increment of disease incidence caused by fungi such as Phytophthora palmivora and Pythium vexans (Drenth and Guest, 2004; Vawdrey et al., 2005).

Phytophthora palmivora is more frequently reported as pathogens in durian. This fungus causes a variety of symptoms such as leaf spot, root rot, stem cancer and fruit rot pre- and post-harvest (Drenth and Guest, 2004; Lee and Lum, 2004; Sivapalan et al., 1997). It has characters that demonstrate it as an effective pathogen, such as: Produces a sporangium which able to survive and spread through the air, rapid sporulation in host plants, its zoospore able to enter the roots chemostatically and electrostatically and able to live a long life inside and outside the host plant (Drenth and Guest, 2004; Kong et al., 2010; Kueh and Khew, 1982). Phytophthora palmivora was reported to have damaged the durian orchard in Penang (Hasan and Siew, 2000) and durian collection in Australia (Zappala et al., 2002). Yield losses due to this defect in Southeast Asian countries are estimated to be around 20-25% (Drenth and Sendall, 2004). Unlikely, only a few informations is relating to the existence and attack of Pythium on durian. Vawdrey et al. (2005) reported the discovery of Pythium vexans along with Phytophthora palmivora on durian orchard in Queensland, Australia. Whilst, Lim (1990) described that the Pythium vexans is potential pathogens in the nursery and mature durian.

In Indonesia, Phytophthora palmivora is only the pathogen reported to be the causes of durian tree-decline (Muryati et al., 2009; Purwantara et al., 2004; Emilda, 2007; Sunarwati et al., 2007). However, information from the field officer and other informal sources indicate a different pathogen has invaded durian. It has characteristics similar to Phytophthora palmivora in general and is called as dry-phytophthora, since it absent of gummosis and remains dry instead of wet stem cancer symptom. This uncertainty may occur due to limited research and observation has been conducted and little is known about the pathogens.

The family Pythiaceae, especially both genus Pythium and Phytophthora have more than 300 described species which being pathogenic on plants and other organism (Van der Plaats-Niterink, 1981; Park et al., 2013). Identification to the species level morphologically requires a high level of expertise, since some similar species share same characteristics lead to misinterpretation (Godfrey et al., 2003; Spies et al., 2011). Certain technique which provides faster and more accurate identification such as molecular tools (Baldauf, 2008; Crous et al., 2003) therefore, should be implemented. For this purpose, a barcoding conserved region, Internal Transcribed Spacer (ITS) of nucleous ribosomal DNA (nrDNA) is frequently used (Cooke and Duncan, 1997; McLeod et al., 2009; Spies et al., 2011). Baldwin et al. (1995) reported that the ITS region has the advantages that support widely used to study phylogeny. This gene has been used for identification of eukaryotes including fungi turn to the intra-species levels (Appiah et al., 2004; Belbahri et al., 2008).

The objectives of this research were (1) To identify molecular characteristic of Pythiaceae associated with durian decline in Indonesia, (2) To identify morphological characteristic of Pythiaceae associated with durian decline in Indonesia and (3) To determine the geographical distribution of Pythiaceae associated with durian decline in Indonesia.

Improving understanding of these areas will provide basic information for the development of integrated disease management for durian decline in the country.

MATERIALS AND METHODS

Sampling and isolates collection: Samples collection was conducted from 2011 to 2014 covered 32 durian fields in 17 provinces in Indonesia. A number of 43 soil, two leaf and one fruit samples were picked up for fungi isolation. The sites are generally a backyard durian field, except for Subang Experimental Farm at Wera which is a germplasm collection field and Sagalaherang orchard which is a private company.

Isolation of the fungi was conducted using fruit baiting method. The soil sample was firstly mixed with sterile water, then put small part of the sample into green sour apples. The inoculated apple was incubated for several days in room temperature until brown-mold occurred surrounding inoculation spot. Pieces of inoculated apple flesh were put onto the Potato-Carrot-Agar (PCA) medium. Monoculture was obtained by excising a peace of mycelium and cultured on PDA medium containing 5 μg pimaricin, 250 μg ampicillin, 10 μg rifampicin and 50 μg hyemexazol (P5ARH), a modification of P5ARPH medium by Jeffer and Martin (1986). Isolation of fungi from leaf and fruit were conducted by put the piece of fungus-attacked organ directly onto PDA medium containing antibiotic P5ARH.

Molecular identification: Molecular identification of Pythiaceae from durian was mainly based on the sequence of the internal transcribed spacers of the ribosomal DNA (ITS-nrDNA). The DNA template isolation and amplification of ITS region was carried out according to the protocol modified by Grunwald et al. (2011) with few modifications. Isolation of DNA was conducted using boiling method as follow: (1) An amount of mycelium as much of 1-2 cm scraped pipette tip along the surface of growing culture was put into PCR tube containing 100 μL dH2O, (2) The tube containing sample was then put floated onto boiled water (at 96-97°C) for 10 min and (3) Lets the water down to room temperature then ready to be use as DNA template. The ITS region was amplified using the universal primers ITS6 (5’-GAA GGT GAA GTC GTA ACA AGG-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’). The PCR reaction mixture was consisted of 10 μL DNA (boiled mycelium), 25 μL PCR mix (KAPA Biosystems), each of 0.4 μM ITS6 and ITS4 primers and 8% DMSO. Amplification was proceed using Applied Biosystem 2720 Thermal Cycler with condition of pre-denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec. Successful amplification was checked by running 5 μL of amplicon and 1 μL loading dye on a 1% agarose gel in 1×TAE buffer at 100 VA for 30 min.

Sequencing and phylogenetic analysis: The PCR products were submitted to 1st BASE Singapore for direct sequencing using Applied Biosystems genetic analyzer with the BigDye® Terminator v3.1 cycle sequencing kit chemistry. Species identification was then conducted by aligning the sequences with deposited sequences data at National Center for Biotechnology Information (NCBI) using Basic Local Alignment Search Tools (BLAST). The first top entries of sequences of the corresponding species in GenBank were downloaded. Phylogeny tree was constructed for all samples sequences and corresponding species, with Rizoctonia solani as rooted outgroup. Sequences were first aligned with ClustalW. Genetic distance was using Tamura-Nei model, whilst the tree was built using Neighbor-Joining method. The bootstrap was generated using 1000 replications. All of the constructing tree processes were conducted using pc-software Geneious R6 and the softwares mention before are available as plug-in.

Colony motif: In order to identify variation of colony motif amongst isolates, observations were conducted at room temperature (22-25°C). A piece of isolate cultures sized 5 mm were grown on PDA medium amended with P5ARH antibiotic formula. The motif was observed on days 3 to 5 upon the isolate growth reached fully the dish.

Sporangium shape and size: To facilitate for comparison the difference of isolate morphologically to support molecular identification, microscopic observations were conducted to identify the shape and size of the sporangium using a light microscope. Initiation of sporangium development was conducted by grown the isolate in sterile soil extract under luminous room for 48 h. A piece of colony was then put on object glass and impressed with lactophenol blue. The observation was then carried out at 400x magnification.

RESULTS

Pythiaceae isolates: Isolation of fungi conducted in the years 2011 to 2014 was successfully obtaining a total of 36 isolates from 32 durian fields. Of those, 34 were baited from soil and each of 1 isolate from leaf and fruit. They represented 17 provinces in five major islands in Indonesia, namely Kalimantan, Sumatra, Jawa, Sulawesi and Papua with the number of 7, 7, 14, 4 and 2 isolates, respectively, as well as two islands of Bangka-Belitung and the Maluku each of 1 isolate (Table 1). A total of 24 isolates obtained from durian fields which were occurring tree-decline incidence and the remaining 12 isolates were not occurring tree-decline incidence.

Molecular characterization: Generated sequences of ITS region using primers ITS6 and ITS4 for 36 isolates separated them into 7 species of Pythiaceae (Table 1). Fifteen isolates showed 98-99% identity to Pythium cucurbitacearum strain 1341pc (HQ237483.1), except for Pp-21, Pp-17 and Pp-33 isolates which showed 88, 95 and 97% identity, respectively. Nine isolates showed 95-98% identity to 5 different strains of Pythium vexans, namely Pythium vexans (GU931701.1), Pythium vexans (JQ898479.1), Pythium vexans Lev3100 (HQ643954.1), Pythium vexans STE-U6728 (GU133594.1) and Pythium vexans WPC3980 (FJ801894.1). Seven isolates showed 94-99% identity to Phytopythium sp., strain AL-2010 (HQ643400.2). Two isolates showed 100% identity to Phytophthora cinnamomi strain parvispora (GU191211.1). Three isolate showed 99, 98 and 99% identity to Phytophthora palmivora (AM422704.1), Pythium sp. D37 (JN863978.1) and Pythium deliense CBS413.33 (AY598674.2), respectively.

Variation of species obtained was not only from difference site but also from the same durian field. Isolates Pp-15 and Pp-37 both were obtained from Wera but they identical to two different species, Pythium sp. D37 and Pythium cucurbitacearum. These two isolates are also different with two other samples from Wera, Pp-43 and Pp-45 which identical to Phytophthora palmivora (AM422704.1) and Pythium delinse (AY598674.2), respectively which both isolated from leaf and fruit host. Similarly, the different species was also obtained from three other locations namely Kaumrejo, Waturejo and Luwu Utara, where each were found two isolates identical to two species Phytopythium sp. (HQ643400.2) and Pythium cucurbitacearum (HQ237483.1).

The parsimoniously phylogenetic tree constructed from the consensus sequence of 36 isolates and their seven corresponding species was forming six clades (Fig. 1), of which two big clades consisted of Pythium cucurbitacearum and Pythium vexans isolates and five small clades consisted of Phytophthora palmivora, Phytophthora cinnamomi, Pythium deliense, Pythium sp., D37 and Pp-19 Krajan isolates. The two large clades are then subdivided into each five smaller clades. The isolates identical to Phytopythium sp., all joined to the clade of Pythium vexans. The corresponding species Phytopythium sp. (HQ643400.2) is very close to Pythium vexans (JQ898479.1), where both stand in the same subclade with bootstrap value of 100. Therefore, it is considered six corresponded species instead of seven as first stated above.

Table 1: Molecular characteristic of Pythium and Phytophthora associated with durian in Indonesia

Fig. 1:
Phylogenetic tree representing the relationship of Pythium and Phytophthora associated with durian in Indonesia. I: Phytophthora palmivora, II: Phytophthora cinnamomi var. parvispora, III: Pythium deliense, IV: Pythium sp., D37, V: Pp-19 Krajan, VI: Pythium cucurbitacearum, VII: Pythium vexans

Amongst the isolates identical to Pythium vexans, Pp-19 Krajan stands a distinct clade outside the groups. This isolate is considered to be different to the six species obtained, or might be a new phylotype since the alignment has only 96% identity (Pryor and Gilbertson, 2000; Liu et al., 2010) with the corresponding species Pythium vexans STE-U6728 (GU133594) and lower identity value with another Pythium vexans strains.

Fig. 2(a-g):
Sporangium shapes of Pythium and Phytophthora associated with durian in Indonesia, (a) Pythium cucurbitacearum, (b) Pythium sp., D37, (c) Phytophthora cinnamomi, (d) Pythium vexans, (e) Pythium deliense, (f) Phytopythium sp. and (g) Phytophthora palmivora

Morphological characterization: Morphological characterizations were conducted through observation of colony motif and sporangia apparent (Table 2). Three basic types of colony motif were found on the isolate culture. Those are petallate, stellate and stoloniferous, each of 20, 15 and 1 isolate, respectively. Petallate motif consists of 3 sub-motifs; they were narrow petallate, medium and wide petallate, each of 4, 14 and 2 isolates, respectively. The stellate consists of 2 sub-motifs namely normal stellate and fluffy stellate, each of 14 and 1 isolate, respectively. Two main motifs, patellate and stellate, were found on the 35 isolates identical to the six species of Pythium and Phytophthora. Whilst, stoloniferous was only found as the motif of isolate Pp-15 Wera.

Sporangiums of the isolates identical to Pythium cucurbitacearum (Fig. 2a) were dominated by globose shape. Of which could also be found another minor shape such as ovoid, ellipsoid, obpyriform, lemon form and irregular.

Table 2: Morphological characteristics of Pythium and Phytophthora associated durian in Indonesia
na: Data not available, +: Papillated sporangium, -: Unpapillated sporangium

The sporangia were generally did not have papillae except for some sporangia which have papillae. In general, the sporangium size of the isolates in this group ranged from the smallest of 7 μm length×5 μm width, to the largest of 25 μm length×22 μm width.

Similar to isolates identical to Pythium cucurbitacearum, the sporangiums of the isolates identical to Pythium vexans (Fig. 2d) were also dominated by globose shape, meanwhile another shape such as ovoid, ellipsoid, oblong, lemon form, obpyriform and irregular were also there in very small number. The sporangia were generally absent of papillae except for a small number of sporangia which were present. In general, the sizes of sporangium of the isolates in this group were ranging from the smallest of 7 μm length×5 μm width, to the largest of 28 μm length×24 μm width. Isolates identical to Phytopythium sp. (Fig. 2f) also have characteristic similar to previous isolate groups Pythium cucurbitacearum and Pythium vexans in shape and the presence or absence of papillae. The sporangium size was ranging from the smallest of 7 μm length×6 μm width to the largest of 23 μm length×22 μm width. Characteristic similarity of the isolates with those identical to Pythium vexans are in conformity with the phylogenetic tree where generally the two groups merge into one clade.

Sporangium shape of isolate identical to Pythium sp. D37 (Fig. 2b) were all globose. Unlike the previous three isolate groups, the only isolates (Pp-15) obtained identical to this species does not have the papillae. It has sporangium width of 15-23 μm which is greater than the three preceding groups.

The sporangia of isolates identical to Pythium deliense (Fig. 2e) were globose and ellipsoidal. They did not have any papillae. The sizes were larger than the average of all Pythium species observed in this research. They were 18-26 μm length and 17-22 μm width. Unique characteristic of this isolate compared to all other isolates observed is that in the ellipsoid sporangium apparently composed of two compartments.

Isolates identical to Phytophthora cinnamomi var., parvispora (Fig. 2c) have sporangium which dominated by globose shape. The other shapes such as ovoid, ellipsoid, oblong and irregular were also found. They did not have any papillae at all. In general, the uniqueness of these isolate is the small size of sporangiums which were about 7-8 μm width or length, except for irregular shapes which were generally longer.

Unlike the six previous groups, the sporangiums of Pp-43 isolate which identical to Phytophthora palmivora (Fig. 2g), were dominated by ovoid and obpyriform shapes. Although other shapes such as globose and irregular also could be found. In general the sporangium seems more prominent because of the relatively larger size with length and width ranging 28-51 and 19-39 μm, respectively. It is 3-5 times fold the size of the sporangium of the other isolates. Another specific characteristic of this isolate is all sporangium has papillae, even though the globose shape.

Geographical distribution: Amongst six corresponding species, two species Pythium cucurbitacearum and Pythium vexans were dominant isolates found association with durian in Indonesia. Of the 17 provinces observed, Pythium cucurbitacearum were found in 13 provinces (76.5%) and Pythium vexans were in 10 provinces (58.8%). Both species were also found to be existed together in six provinces. Phytophthora cinnamomi were found in two provinces, whilst another three species, Phytophthora palmivora, Pythium deliense and Pythium sp., D37 were only found in one location (Fig. 3).

Fig. 3: Distribution maps of Pythium and Phytophthora associated with durian in Indonesia

DISCUSSION

Tree decline is a serious disease of durian tree in Indonesia. However, due to the culture practices is still dominated by non-intensive cultivation; the disease has not been widely perceived by the growers. This disease is almost related to Phytophthora palmivora as the responsible pathogen (Muryati et al., 2009; Purwantara et al., 2004; Emilda, 2007; Sunarwati et al., 2007). Meanwhile, Vawdrey et al. (2005) reported the Pythium vexans also found associated with durian tree-decline.

Recent development of durian cultivation in Indonesia which is starting to shift from subsistence towards intensive culture practices need to prepare disease management control. Because of intensive agriculture practices usually lead to increase disease problems (Drenth and Guest, 2004). At the beginning is necessary to identify the pathogen accurately and its distribution to create more specific and effective strategic control.

In this study, a total of 36 Pythiaceae isolates were found associated with durian in Indonesia. These isolates were found both in the location that there is disease incidence and no incidence at the time of collection. The presence of pathogens in both types of locations is indicating that the fungi are able to survive in the soil for long time. Vawdrey et al. (2005) reported that two species of Pythiaceae, Phytophthora palmivora and Pythium vexans were found in two climatic conditions, wet and dry, however no information how long these fungi can survive in the soil. As comparison, Fusarium oxysporum (foc) that attack banana can survive for 30 years in the soil without a host (Ploetz, 2006). Further research to identify how long they could survive in the soil is important to diseases management.

Molecular identification using ITS sequences found all 36 isolates correspond to six Phythiaceae species: Pythium cucurbitacearum, Pythium vexans, Phytophthora cinnamomi, Phytophthora palmivora, Pythium sp., D37 and Pythium deliense. Two species Pythium cucurbitacearum and Pythium vexans consisted of ten distinct strains or phylotypes. This is more numerous than those found by Vawdrey et al. (2005) in Queensland, Australia which found Phytophthora palmivora and Pythium vexans. Variations which arise at the species level and at strain or phylotype are not surprisingly, because this country is known as one of the mega-biodiversity in the world. Beside as the center of origin and diversity of the genus Durio (Kostermans, 1958; Brown, 1997), Indonesia also grows many other potential hosts for Pythiaceae. For a long time this condition, therefore, could emerge new pathogen species as a result of reciprocated interaction between them (Frank, 1992).

The diversity is also indicated by the variation in colony motifs. There are six motifs were found which is slightly different with that found by Pongpisutta and Sangchote (2004). However, comparison amongst colony motifs and molecular identification results seem no correlation were found. Unlike Pp-15 isolate (correspond to Pythium sp., D37) from Wera which has the only stoloniferous motif, another isolates might have a stellate or patellate motif even though they are Pythium sp., or Phytophthora sp. Based on this finding, it was found that the motive colony could not be used as an identifier to distinguish between Phytophthora and Pythium species.

Among the isolates observed, it was generally found six types of sporangium, those are globose, ovoid, ellipsoid, oblong, lemon form and obpyriform, as found by Pongpisutta and Sangchote (2004) on the isolate of Phytophthora palmivora from durian. However, there is prominent variation in the size of sporangium found among isolates in this study (Fig. 2). Isolates in a group of identical species generally have diverse sporangium and almost all the basic shapes are there in the same identical group. It leads difficulty to differentiate morphologically. Amongst inter-species isolates are generally also difficult to distinguish. Such isolates identical to the two species, Pythium cucurbitacearum and Pythium vexans, they share identical characteristics of sporangium shape and size. However, by using the molecular tools such as ITS region was able to distinguish amongst the species (Appiah et al., 2004; Belbahri et al., 2008), even though at lower taxa as shown by the phylogenetic tree (Fig. 1).

Isolate Pp-43 which identical to Phytophthora palmivora is only obtained from leaf samples, while Pp-45 which identical to Pythium deliense is from the fruit. Both are different from another 34 isolates were obtained from the soil, even though from two isolates obtained from the same location (Pp-15 and Pp-37). It is, therefore, assumed that each fungus has different preferences on plant organs, or they have different modes of transmission. Phytophthora palmivora is able to survive and spread through the air (Drent and Guest 2004), is a reasonable answer for the finding isolate Pp-43 from the leave.

The present study demonstrated the first publication for the association of Pythium cucurbitacearum with tree-decline of durian. Pythium cucurbitacearum together with Pythium vexans provide evidence their present throughout the region observed (Fig. 3). Based on these findings we could infer that pathogen that attack durian which called as the dry-Phytophthora is referring to the Phythium species, it could be Pythium vexans or Pythium cucurbitacearum, as both species are more commonly found in most of the soil samples compared to Phytophthora. This is supported by the symptom found on the durian stem which showed absent of gummosis or wet cancer (Thompson, 1934, 1938).

ACKNOWLEDGMENT

This study was funded by Indonesian Agency for Agricultural Research and Development through program KKP3T Fiscal Year 2011-2013.

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