Fakultas Pertanian dan Bisnis Universitas Kristen Satya Wacana Jl. Diponegoro 52-60 SALATIGA 50711 - Telp. 0298-321212 ext 354 email:[email protected], website: ejournal.uksw.edu/agric
Terakreditasi Kementrian Riset, Teknologi dan Pendidikan Tinggi berdasarkan SK No 200/M/KPT/2020
Received: 5 July 2021 | Accepted: 10 February 2022
EVALUATION OF LOCALLY RICE SEEDS HEALTH Tri Martini*, Evy Pujiastuti*, Siti Nurhaeni, Mekky Kusuma Dewi,
Nur Indrayati Praba Ningrum, dan Mansyur
* Balai Pengkajian Teknologi Pertanian (BPTP) Yogyakarta
Balai Besar Pengembangan dan Pengujian Mutu Benih TPH (BBPPMBTPH) e-mail: [email protected] , [email protected]
ABSTRACT
Seed health is one of the important factors in maintaining seed quality. This factor is very influential both during seed storage, plant growth in the field, and production to be produced.
Seed can be one of the effective media in the spread of plant diseases. With the consideration that pathogenic seed transmission plays an important role in the spread and development of epidemic diseases in some commodities, then gradually seed health tests need to be incorporated into the seed certification process. Seed health testing is required to detect the presence of pathogens or seed health status. To support the program of activities of the Ministry of Agriculture in the development of local rice, it is necessary to start by evaluating the health of seeds in the local paddy varieties. The purpose of the research includes detecting the type of pathogens carried by local varieties in several provinces and inventories of pathogen data carried by local varieties of rice seeds circulating in several provinces. The research method was conducted by taking samples directly from the field and conducting DNA isolation by PCR test to detect bacteria Xanthomonas oryzae pv oryzae (Xoo) and Burkholderia glumae (BG) as well as the growth of pathogen samples in the laboratory. The results of identification are known some seeds detected bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Burkholderia glumae (BG)) which is characterized by the emergence of DNA bands PCR amplification results in positive control according to DNA Ladder. The results of the identification of pathogenic fungi, known the presence of fungi causing blast disease (Pyricularia oryzae) in the overall example of rice seeds tested (47 examples of local rice seeds upland and local swamp) and there are 15 examples or 32% infected with a percentage of infections 0.25 - 14.75%. While result identification of nematodes, known the presence of parasitic nematode Aphelenchoides besseyi on the overall sample of local rice seeds tested (42 examples of local rice seeds upland and local swamp), with 25 examples or 59.5% infected with an infection range of 1-44 specimens.
Keywords: identification, health, seeds, rice, varieties, local
INTRODUCTION
Climate change due to global warming over time is an increasingly visible impact on all joints of life. Impacts that have been felt today include extreme weather changes, rising temperatures, rising sea levels, changes in extreme rainfall intensity, seasonal shifts, and decreased crop yields. Because the impact of climate change occurs in all sectors, including agriculture, one of the strategies that must be done is adaptation.
To face global competition, especially entering the era of the ASEAN Economic Community, it is necessary to increase competitiveness so that the agribusiness industry in the country continues to develop.
The right agricultural production system adaptation strategy for a very large area and different climate environments such as Indonesia is the need to develop strategies for the short and long term. The development of new varieties that can adapt takes a short time with a percentage of success that is not necessarily as expected. Meanwhile, the provision of food for the people cannot be delayed because it is prone to instability in the life of the nation and state. To anticipate the occurrence of this, the selection of a strategic short-term strategy is to develop local varieties (Supangkat, 2017).
The existence of local rice varieties is very strategic in the efforts to fulfill food in the future, in addition to adaptive superior varieties that are still in the process of discovery or discovery (invention). Some local varieties have the genetic advantage to be used as a source of genes in the assembly of new superior varieties, both their tolerance to iron poisoning, the resistance of some soil diseases, and the high Fe and Zn content in their rice (Khairullah, 2016). But according toKoesrini et al. 2014 in Darsani
and Koesrini (2018), the yield of local varieties is low (2.0-2.5 t/ha), longevity (8-10 months), and relatively impervious to pests and plant diseases. Breeding activities are a strategic step to increase competitiveness by creating superior varieties that are adaptive to climate change, resistant to pests/diseases, and under consumer preferences.
Although local varieties have disadvantages compared to nationally reputable varieties that are short-lived (about 4 months), and high yields (about 7-10 t/ha), farmers to date still survive working on local varieties. These conditions need to be studied to find out the extent of the competitiveness / competitive advantage of local varieties of rice compared to the competitiveness of national superior rice varieties from social and economic aspects. Based on the above problems, the study of the competitive advantage of local varieties against national superior varieties is crucial because it is not in line with the previous national production increase program that directs farmers to grow national superior varieties.
Seed health is one of the important factors in maintaining the quality of the seed. This factor is very influential both during seed storage, plant growth in the field, and the production that will be produced. In addition, seeds are one of the effective media in the spread of plant diseases.
Considering that seed-borne pathogens play an important role in the spread and development of epidemic diseases in some commodities (Agrios, 2005) then gradually seed health tests need to be incorporated into the seed certification process.
Seed health testing is necessary to detect the presence of pathogens or seed health status.
As one of the activities to support the development of local varieties of rice seeds, it is necessary to pay for disease resistance of local
rice varieties, especially for local upland rice and local swamps. The purpose of the study was to detect pathogenic types and inventory pathogen data carried by local varieties of rice seeds circulating in several provinces.
RESEARCH METHODS
The activity was held from January to December 2019 at the Microbiology Laboratory of Assessment Center on Development and Testing of Food Crop Seeds and Horticulture (Balai Besar Pengembangan dan Pengujian Mutu Benih Tanaman Pangan dan Hortikultura / BBPPMB-TPH), Director General of Food Crops, Ministry of Agriculture.
Pada testing used materials in the form of local swamp rice seeds and local upland rice was taken from 3 provinces where local rice development sites are Jambi, South Sumatra, and South Kalimantan. Research methods used for pathogen detection using standard testing include 1) blotter test methods for fungi testing;
2) PCR methods for bacterial testing; and 3) ISTA (International Seed Testing Association) method, 2016 for nematodes testing.
Supporting fluids are used such as autoclave, laminar airflow, stereo microscope, compound microscope, incubator, petri dish, sprayer, bunsen, tweezers, thread tubes, Eppendorf, micropipette, centrifugation, and others. The specific primers used are 5'- GCATGACGTCATCGTCCTGT-3' forforward and 5'-CTCGGAGCTATATGCCGTGC-3' for reverse with a product length of 470 bp. PCR runs 29 cycles with predenaturation stages at
95oC for 2 minutes, then denaturation (DNA thread separation phase) at95oC for 30 seconds, 63oC temperature for 30 seconds for annealing (primary integration), extension (synthesis of new DNA strands) at 72oC for 1
minute, continued the post extension stage of 72oC for 7 minutes. For colony, B. glumae (BG) used a DNA template obtained from the results of subsequent isolation entered the PCR process. PCR is done using a primer specific to BG. The specific primers used are 5’
TGGGTAGTCTCTGTAGGGAA-3' for forwardand 5'-TCATCCTCTGACTGGCTCAA- 3' for reverse with a product length of 164 bp.
PCR runs 44 cycles with predenaturation stages at 94oC for 3 minutes, then denaturation (DNA thread separation phase) at 94oC for 30 seconds, 58oC temperature for 30 seconds for annealing (primary integration), extension (synthesis of new DNA strands) at 72oC for 30 seconds, then continued post-stage Extention 72oC for 10 minutes (BBPPMBTPH, 2018).
RESULTS AND DISCUSSIONS
- Seed Sampling and Observation of Disease Symptoms in the Field
Seed sampling and direct observation of potential disease symptoms that appear are carried out in several local rice field locations simultaneously or close to harvest time in each of the target provinces namely Jambi, South Sumatra, and South Kalimantan. Seed sampling is intendedto obtain the seed source used in the health evaluation activities of local rice seeds.
From the observation of visually seen symptoms of the disease that appeared in several locations of local upland rice fields in three districts of Jambi Province (Muara Jambi, Sarolangun, and Tebo) that showed symptoms of blast disease attacks. Similarly, observations in some local swamp rice fields in Banyuasin and Ogan Komering Ilir (OKI) regencies in South Sumatra Province, as well as in some lands in Tanah Laut and Banjar regencies in South Kalimantan
Province (Kalsel)also showed symptoms of blast and crepe disease. In Figures 1 and 2 can be seen the results of documentation of observation of disease symptoms at the time of seed sampling activities in the field.
At the time of observation, no signs or symptoms of other diseases were noticeable other than blasts such as crepe disease, rotten bullies, and white shoots. However, this needs to be further confirmed through laboratory testing. The local variety of rice seeds that are
Figure 2 A sampling of seeds with spotting on rice fields in South Sumatera and South Kalimantan provinces
Figure 1 Symptoms of blast disease at the local upland rice field in Merangin-Jambi
widely developed in the three provinces are generally unregistered and have a long harvest life of more than 4.5 months (135 days). The sampling of seeds other than in the three provinces is also carried out in Central Kalimantan Province for seeds that have been harvested for several days. Examples of seeds that have been taken from the field are then cooled and homogenized (mixed into 1 part/
container evenly). Furthermore, the test was carried out at the Laboratorium of microbiology
to detect the presence of seed-borne pathogens.
Testing is intended to check if there are potential attacks of disease in the field such as blast, blight, brown leaf spot, BLB (Bacterial Leaf Blight), striped leaf patches, BLS (Bacterial Leaf Streak), and bakanae.
- Seed Health Testing in the Laboratory 1. Test results of fungus and Nematoda Hasil fungi tests conducted on 47 examples of seeds taken from 4 provinces (Table 2) showed the existence of examples of local varieties of rice seeds in Jambi and South Sumatra provinces that showed attacks of blast disease although with low infection rates ranging from 0.25 - 14.75%. Especially in Kalsel Province, when direct observations are made in rice fields there are symptoms of blast disease, but allegedly because the level of attack is relatively mild when tested in the laboratory the target fungi is not detected.
Meanwhile, in the results of nematodes tests detected A. besseyi infection in seed samples from Jambi, South Sumatra, and Center Kalimantan provinces, although it is known that the infection rate is still relatively mild, ranging from 1-44 specimens. According to Aziz (2013), brown spot on leaf smelt is rare. The distinctive shape of the leaf blast patches is quadrangular with two more pointed ends.
While the symptoms caused in the field due to the attack of nematodes A. besseyi include the tip of the leaves becoming white, twisted, malai becoming lunglai, while in the bullion there are uneven brown patches, and hollow (Lubis, 2017).
Following Table 1 49 local rice varieties have been taken from. Data in Table 1 and Diagram Figure 3 on the comparison of the results of the fungi and nematodes tests in the four provinces,
showed that the results of fungi tests that had been conducted on 18 examples of local rice seeds from Jambi Province found 50% of seed samples detected by P. oryzae and nematodes test results on 17 examples of seeds there are 76% sample seeds detected the presence of A. besseyi. This is by the assumption that Pyricularia is a dynamic pathogen and can adapt quickly to the conditions of the host plant.
Pathogens also have a high degree of genetic diversity and the ability to produce new breeds (Nasution & Usyati, 2015). The results of tests on 7 examples of local swamp rice seeds from South Kalimantan Province were not detected in the presence of fungus P. oryzae and A.
besseyi. Meanwhile, the sample of fungi tests on 17 examples of local swamp rice seeds from South Sumatra Province contained 35% of the examples of seeds detected by P. oryzae and nematodes test results on 15 seed samples there are 53% of examples of seeds detected A.
besseyi. Test results fungus on 5 examples of local swamp rice seeds from Center Kalimantan Province has not detected the presence of P. oryzae, but the results of nematodes tests on 4 examples of seeds tested 100% detected the presence of A. besseyi.
Following Table 1 49 local rice varieties have been taken from. Data in Table 1 and Diagram Figure 3 on the comparison of the results of the fungi and nematodes tests in the four provinces, showed that the results of fungi tests that had been conducted on 18 examples of local rice seeds from Jambi Province found 50% of seed samples detected by P. oryzae and nematodes test results on 17 examples of seeds there are 76% sample seeds detected the presence of A. besseyi. This is by the assumption that Pyricularia is a dynamic pathogen and can adapt quickly to the conditions of the host plant.
Table 1 Data from the test results of P. Oryzae fungus and nematode A. besseyi on local varieties of rice seeds
No. Types of Plants Varieties
Identification Results P. oryzae (%) A. besseyi
(Spesimen) 1
Rice Fields/Upland rice from Local
Jambi
Seni Emas 0 0
2 Seni Lentik 0 -
3 Seribu Naik 1 2.75 2
4 Seni 0 5
5 Seni Sasak 2.00 0
6 Marinai 1.00 5
7 Kunangan 0 0
8 Kuning 14.75 16
9 Sikuning 0 1
10 Tunggung 4.75 0
11 Seni Mungin Putih 7.00 27
12 Kasa 0.25 3
13 Sungkai 5.00 7
14 Perak 0 2
15 Sialas 0 2
16 Simerah 0 2
17 Seribu Naik 2 0.25 7
18 Kuku Balam 0 31
% Infection from the Number of Seeds Taken 50% 76%
19
Swam local rice from South Kalimantan
Siam Udi 0 0
20 Rukut 0 0
21 Siam Mayang 0 0
22 Siam Cantik 0 0
23 Siam Rantau 0 0
24 Siam Pandak 0 0
25 Siam Unus Kuning - -
26 Mayang 0 0
27 Ketan Siam - -
% Infection from the Number of Seeds Taken 0% 0%
28
Swam local rice from South
Sumatera
Melati 1.00 0
29 Sulaiman 0 -
30 Makmur 0 13
31 Bromo 0 3
32 TW 0 0
33 Kuda 0 0
34 Makmur 16 1.25 0
35 Merah Jao 0 0
36 Makmur 8 0 -
37 Sungkai 0 9
38 Vietnam 1 0 2
39 Sulaiman 18 0.75 4
40 Makmur 15 0.75 11
41 Kuku Balam 0.0 0
42 Sulaiman 19 0.75 30
43 Sulaiman 20 0.50 0
44 Vietnam 2 0 27
% Infection from the Number of Seeds Taken 35% 53%
45
Swam local rice from South Kalimantan
Raden Ranah 0 -
46 Parukat (Samer) 0 44
47 Palut 0 9
48 Pandan Wangi 0 5
49 Dite Dangkak 0 11
% infection from the number of seeds taken 0% 100%
Sign (-) = No testing data
Pathogens also have a high degree of genetic diversity and the ability to produce new breeds (Nasution & Usyati, 2015). The results of tests on 7 examples of local swamp rice seeds from South Kalimantan Province were not detected in the presence of fungus P. oryzae and A.
besseyi. Meanwhile, the sample of fungi tests on 17 examples of local swamp rice seeds from South Sumatra Province contained 35% of the examples of seeds detected by P. oryzae and nematodes test results on 15 seed samples there are 53% of examples of seeds detected A.
besseyi. Test results fungus on 5 examples of local swamp rice seeds from Center Kalimantan Province has not detected the presence of P. oryzae, but the results of
nematodes tests on 4 examples of seeds tested 100% detected the presence of A. besseyi.
The results of detection and identification of fungi carried out on all examples of seeds show the presence of various types of fungus other than target fungi (Pyricularia oryzae) among others:
Acremonium sp., Alternaria spp. , Aspergillus spp. , Bipolaris sp., Cercospora sp., Chaetomium sp., Cladosporium sp., Curvularia sp., Fusarium spp., Gelarchia oryzae, Nigrospora sp, Penicillium sp., Phoma sp., Rhizopus sp., Tilletia barclayana., Trichothecium sp., and Trichoderma, sp. In Figure 4 can be seen the morphology of the fungi P. oryzae and nematodes A. besseyi under a compound microscope.
P. oryzae
A. besseyi
Local Upland Rice of Jambi
Local Swamp Rice of South Kalimantan
Local Swamp Rice of South Kalimantan Local Upland
Rice of South Sumatera
Figure 3 Percentage of the number of local rice seed samples detected and undetected by fungi and nematodes from all the examples tested
Figure 4 Microscopic identification results; a. Morphology of P. oryzae;
a b
Stilet
Konidia
Konidiofor
b. Morphology of A. besseyi
The detection of P. oryzae (Figure 4a) is shown by the characteristic of morphology in the form of straight or notched conidiophores, forming elbows at the ends, light brown can reach 200 ìm in length, 3-5 ìm wide. Conidia clustered, egg-shaped with pointy ends, obpyriform, obclavate, hyaline to light brown, generally consisting of 2 septa, conidia size 17-30 x 7-10 ìm. (Ik-Hwa Hyun, 2004). Morphology of A.
besseyi found slender, cylindrical, and body position at the time of active or life moving curved and stilet with stomata stilet type (Figure 4b).
Testing bacteria with PCR techniques The testing of seed-borne bacteria with the target of Xanthomonas oryzae pv oryzae (Xoo) and Burkholderia glumae (BG) bacteria was carried out on 2 examples of seeds of local varieties Makmur and Makmur 15 from South Sumatra Province consisting of several seed grains with bacterial colonies that were included in the tube as a sub-sample. The initial stage required for testing with PCR techniques is an easy and practical DNA isolation method (Fatmawati, 2017). The total DNA extraction
of Xoo and BG bacteria is obtained from the isolation of bacterial DNA grown in common breeding media namely Potato Dextrose Agar (PDA) media and Xanthomonas Agar (XA) selective media for Xoo. The choice of PDA and XA media use is because the media is relatively simple in the manufacturing process.
But to growing colonies of Xoo and BG bacteria on PDA media is still hampered by the growth of seed-carried fungi in the media so in the process of DNA isolation it is difficult to get pure bacterial DNA. This is thought to be because the sterilization treatment of seed surfaces using a 1% Clorox solution for 1 minute is not optimal enough to suppress the growth of fungi. So for the next test, it is necessary to sterilize the surface by increasing the concentration of Clorox solution 3% for 1-2 minutes or the use of a type of antibiotic at the time of making the media.
In the early stages of DNA isolation of Xoo and BG bacteria using the Schaad (Schaad., et al. 2001) method, white bacterial colonies were taken in 2 samples of local rice seed varieties bred on PDA media (Figure 5-6).
Figure 5 Growth of bacterial colonies on PDA media contaminated with fungi
a. Xoo detection with PCR techniques DNA templates obtained from the isolation of yellow bacterial colonies on PDA media are then carried out PCR process. Pcr results are seen in Figure 6, which shows Hasil PCR DNA Xoo bacteria with DNA template without dilution. Hasil test uses Xoo positive control, no DNA band of the same sample as DNA ladder band or positive control so the test results for all samples are not detected Xoo bacteria.
Although isolation methods can be used, PCR testing of bacterial DNA taken from colonies grown on PDA-in order media still requires optimizing DNA electrophoresis programs so that ladder separation can be seen more clearly.
Figure 6 PCR results in Xoo bacterial DNA with unluted template DNA from bacterial colonies in PDA media (1=100bp DNA ladder; 2, 10, 18, 26 = positive control; 3-8, 11-16, 19-24, 27-32 = isolation of Xoo
DNA from bacterial colonies in PDA media)
b. Detection of BG with PCR techniques Colonies of B. glumae (BG) on PDA media are grayish-white. The DNA template obtained from the results of the isolation is further entered in the PCR process. PCR results are seen in Figure 7 where the DNA results of BG bacteria are positive with DNA templates without being diluted.
In the PCR process, four positive controls of Xoo and BG bacterial isolates were used. The PCR process was declared successfully characterized by the appearance of PCR amplification DNA bands on all four positive controls, although in positive control BG (column 18) was not perfectly identified. Of
Figure 7 PCR results of BG bacterial DNA with unluted template DNA (1=100bp DNA ladder;
2,10, 18, 26 = positive control; 3-8, 11-16, 19-24, 27-32 = isolation of BG DNA from bacterial colonies in PDA media)
the same 24 samples tested with primary Xoo and BG (Figures 6 and 7) showed the same results as DNA Ladder and BG positive control DNA bands at 164 bp: columns 3-15, 19-24, and 27-32. Based on PCR results on two local varieties, namely Makmur and Makmur 15 which were tested with 2 primers (Xoo and BG) in both varieties only detected BG bacteria.
CONCLUSIONS
1. The results of the identification of fungi that cause blast disease (Pyricularia oryzae) is the discovery of as many as 15 out of 47 examples of local rice seeds upland and local swamps tested () or 32% of infected samples with a percentage of infection 0.25 - 14.75%;
2. Hasil identification of parasitic nematodes Aphelenchoides besseyi is the discovery
Testing Methods. International Seed Testing Association (ISTA), Bassersdorf, Switzerland.
Ik-Hwa Hyun. 2004. Identification of Three Fungi Newly Intercepted from Importing Plants in Korea. Journal of Mycobiology 3 3 ( 4 ) : 2 4 3 - 4 . D O I : 1 0 . 4 4 8 9 / MYCO.2005.33.4.243
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Lubis, L.A., 2017. Deteksi Aphelenchoides Besseyi pada Berbagai Varietas Padi (Oryza Satival) di Sumatera Utara.
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Schaad, N.W, Jones J.B. and Chun W.
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of as many as 25 out of 42 examples of local rice seeds (upland and swamp) tested 25 or 59.5% of infected samples with an infection range of 1-44 specimens;
3. Tests with the PCR method can detect Xanthomonas oryzae pv oryzae (Xoo) and Burkholderia glumae (BG) bacteria characterized by the appearance of PCR amplification DNA bands on positive control according to DNA Ladder.
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