JP3413490B2 - Microbial infection diagnosis method - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for diagnosing microbial infection for diagnosing the presence or absence of Ganoderma infection on oil palm. [0002] Oil palm has been known as a major oil and vegetable resource in tropical countries such as Malaysia and Indonesia. Oil palm vegetable oil productivity is 4
It is as high as ~ 7 tons / ha year, and the world's total production is the second largest in the world after soybean oil. About 50% of this is produced by Malaysia and about 20% by Indonesia. [0003] In these tropical countries,
Basal Stem Ro
t) is a problem of oil palm soil disease.
Basal stem lot is a disease peculiar to oil palm that begins with root infection by a soil microorganism, Ganodermaboninense, and eventually causes the affected part to spread from the root to the stem and the leaves to die. Basal
Stem lot is characterized by symptoms appearing late in infection,
At that point, about 50% or more of the tree tissue at the edge of the stem has been violated. Therefore, conventionally, even if a disease infection is detected by finding a symptom, at the time when the infection is detected, about 50% or more of the tree tissue at the stalk base is violated. [0004] Therefore, even if an infection is detected, drug administration or surgical treatment is not an effective treatment in a state where about 50% or more of the tree tissue at the edge of the stem is violated. . For this reason, when an infection is detected, the disease has already progressed, and the production of fats and oils is greatly reduced. Also, when an infection is detected, the tree will eventually die because no effective treatment can be given. For this reason, even if an infection is detected, economic loss is a serious problem. Further, when removing diseased trees in which infection has been detected or dead trees in which infection has completely progressed from the plantation, diseased tissues such as roots cannot be completely removed from the plantation. You may get infected. Therefore, the number of trees affected by the basal stem lot tends to increase year by year. Therefore, there is a need for a new diagnostic method for quickly and easily diagnosing whether or not an oil palm is affected by a basal stem lot. That is,
By quickly and easily detecting whether or not a given oil palm is infected with a genus Ganoderma, the above-described economical loss can be reliably prevented. [0007] From such a viewpoint, when no clear symptom can be found yet, the root or stem margin is drilled to collect the tree tissue, and the color of the browned tissue piece and the selective medium of the genus Ganoderma are selected. A method for diagnosing infection by culturing a collected tissue piece and confirming the appearance of the bacterium has been proposed. However, the inhomogeneity of the collected tissue and the instability of detection caused by the culture operation of the bacteria cannot be overcome, the burden on the plant individual is large due to drilling, and the infection is considerably advanced at the time of infection diagnosis. For these reasons, it is not a technique that achieved early detection of infection. Further, it has been reported that the amount of magnesium ions increases due to infection with Ganoderma (PDTurner,
Poon Yew Chin, "Effects of Ganoderma infection on
theinorganic nutrient status of oil palm tissues "
Oleagineux, 23 (6), 367-370 (1968)), and based on this report, a method of diagnosing infection based on the amount of magnesium ion is also conceivable. However, findings that neglect the accumulation of magnesium ions due to infection with Ganoderma species (Umar Akbar, M. Kusnadi, M. Ollagnier, "Influenc
e of the type of planting material and of mineral
nutrition onoil palm stem rot due to Ganoderma "Ol
eagineux, 26 (8-9), 527-534 (1971)), and the diagnosis of infection by the amount of magnesium ion has not been established from the viewpoint of reliability. SUMMARY OF THE INVENTION Accordingly, the present invention has been made in view of the above-mentioned situation, and has been made in consideration of the above circumstances. It is intended to provide a way. Means for Solving the Problems The present inventors have made intensive studies in order to achieve the above-mentioned object, and as a result, when oil palm is infected with Ganoderma spp. They found that the amount of ornithine changed, and completed the method for diagnosing microbial infection of the present invention. That is, the method for diagnosing microbial infection according to the present invention performs an acid ninhydrin reaction using oil palm leaves as a sample, and measures the amount of amino acids having an absorption maximum near 515 nm in the obtained reaction product, thereby obtaining a ganoderma. It is characterized by detecting infection of a genus Bacteria. Hereinafter, the present invention will be described in detail.
The method for diagnosing microbial infection according to the present invention is a technique for detecting the presence or absence of Ganoderma infection on oil palm. Examples of the genus Ganoderma include Ganoderma boninense. The genus Ganoderma, such as Ganoderma boninens, is a soil microorganism that infects the roots of oil palm, and thereafter from root to stem. And
This ganoderma spawns a disease called the basal stem lot. In the method for diagnosing microbial infection according to the present invention, first, the leaves of the oil palm to be diagnosed are collected, and the leaves are extracted with an organic solvent such as ethanol. The extract obtained by this extraction is subjected to an acidic ninhydrin reaction. At this time, it is preferable that the leaf to be collected is the newest leaf in the oil palm individual to be diagnosed. By using the newest leaves, the physiological conditions in the diagnostic method can be kept constant. The acidic ninhydrin reaction is carried out by a general method,
That is, the ninhydrin reagent (triketohydrindene hydrate) and the above-mentioned extract are mixed under acidic conditions, and the reaction is carried out at a predetermined temperature. According to the acidic ninhydrin reaction, the extract is colored by the reaction between the free amino acid contained in the extract and the ninhydrin reagent. By the way, when the oil palm is infected with the genus Ganoderma, compared to the case where the oil palm is not infected with the genus Ganoderma, a larger amount of amino acids such as proline and ornithine are contained in the oil palm leaves. accumulate. For this reason, the extract of the oil palm leaf infected with the genus Ganoderma exhibits a stronger color due to proline and ornithine in the acidic ninhydrin reaction as compared with the case where the extract is not infected with the genus Ganoderma. When not infected with Ganoderma, oil palm leaves contain almost constant amounts of proline and ornithine. Specifically, a compound having an absorption maximum for light having a wavelength of about 515 nm is produced in the extract of oil palm leaves by the reaction of proline or ornithine with a ninhydrin reagent. Therefore, the amount of proline or ornithine in the extract can be detected by measuring the absorbance of the extract after the ninhydrin reaction using light having a wavelength of about 515 nm. In this method, first, as a control,
The amounts of proline and ornithine in oil palms not infected with the genus Ganoderma are measured by acid ninhydrin reaction. In this method, the amounts of proline and ornithine in the leaves of the oil palm to be diagnosed are measured by the acid ninhydrin reaction, and if there is a significant difference compared to the control value, the oil palm to be diagnosed is Ganoderma. Diagnose that the bacterium is infected. Also,
If the amounts of proline and ornithine in the oil palm to be diagnosed are not significantly different from the control values, it is diagnosed that the oil palm to be diagnosed is not infected with Ganoderma. As described above, according to the present method, the presence or absence of Ganoderma infection on oil palm can be reliably diagnosed based on the amounts of proline and ornithine due to the acidic ninhydrin reaction. In particular, in this method, it is only necessary to collect the leaves of the oil palm to be diagnosed, and damage to the oil palm to be diagnosed can be prevented. Further, according to the present method, it is possible to diagnose the presence or absence of infection at a stage where no symptom due to the infection of the genus Ganoderma is found on the leaves, stems and roots of the oil palm. That is, according to the present method, the presence or absence of Ganoderma infection to oil palm can be diagnosed relatively early. In this method, since the newest leaf of the oil palm to be diagnosed is used, diagnosis is performed under physiologically constant conditions. That is, in the present method, the newest leaf is used for the oil palm measured as a control, and the newest leaf is used for the oil palm to be measured, so that the comparison can be performed in a physiologically equal state. Furthermore, this technique is different from a technique of diagnosing the presence or absence of infection by culturing Ganoderma spp. From a part of the diagnosis target, and can quickly and reliably diagnose the presence or absence of infection. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples. [Example 1] Inoculation of Ganoderma spp. In this example, Ganoderma boninens was inoculated to oil palm seedlings as Ganoderma spp. This Ganoderma Boninens was provided by the Ministry of Agriculture, Forestry and Fisheries Genetic Resource Center to
Stored as FF305601. As the inoculation method, a so-called "rubber wood block" method was employed. That is, first, a piece of rubber wood (6 × 6 × 12 cm) and a 3% malt extract solution are prepared, and the wood piece and the 3% malt extract solution are mixed in a polypropylene container at 121 ° C. for 30 minutes. Sterilization was performed for minutes. Thereafter, Ganoderma boninens was inoculated on the wood pieces, and cultured for 2 months to obtain Ganoderma boninens.
A piece of wood infected with Boninens was obtained. Oil palm seedlings were obtained by growing seedlings germinated from fruits of Tenera species commercially available in Malaysia for 3 months in a small polybag containing soil. Next, the roots of the oil palm seedlings are exposed so as not to be damaged. Then, a piece of wood infected with Ganoderma boninens is placed on the soil filled to a height of 1/3 of the polybac, and the wood piece and the root are brought into contact with each other to fill the remaining space with the soil. Replant the oil palm seedlings. As a result, oil palm seedlings are inoculated with Ganoderma boninens. As described above, even when Ganoderma boninens is inoculated without damaging the oil palm seedlings, not all oil palm seedlings are necessarily infected. Therefore, in the following examples, oil palm seedlings infected with Ganoderma boninens were compared with oil palm seedlings that were not infected (both of which show no symptom in the shoot aerial part and leaves). Example 2 Measurement of Amino Acid Concentration by Acidic Ninhydrin Reaction The measurement of amino acid concentration by acid ninhydrin reaction was carried out by collecting the newest leaves of oil palm seedlings 10 months after inoculation and using the Bates method (Bates, LS, R.
P. Waldren, IDTeare, Rapid determination of free
proline for water-stress studies.Plant and Soil 3
9,205-207 (1973)). Specifically, first, the collected leaves were immediately ground in liquid nitrogen, weighed, and extracted with 5% acetic acid / 25% ethanol. Then, the extract was filtered to remove solids, and then made up to a constant volume with an extraction solvent. 2 mL of the ninhydrin reagent (2.5 g of ninhydrin dissolved in 100 mL of 6 M phosphoric acid / acetic acid) and 2 mL of acetic acid Was added and stirred and heated at 90 ° C. for 1 hour. After heating, the mixture was cooled with water, 4 mL of toluene was added to the reaction mixture, and the reaction product was distributed. The absorbance of the toluene phase at 515 nm was measured.
The analysis of proline and ornithine under the conditions described above was performed by Chinard, FP, Photometric estimation of prol.
ine and ornithine. J. Biol. Chem. 199, 91-95 (195
It is described in 2). In measuring the absorbance, an acidic ninhydrin reaction is performed using proline as an external standard substance, and a calibration curve showing the relationship between the absorbance at 515 nm light and the amount of proline as shown in FIG. 1 is obtained. (Results) The measured value of the absorbance is converted to the amount of proline from the calibration curve shown in FIG. 1 obtained using proline as an external standard substance. Table 1 shows the results of conversion of the amount of proline between oil palm seedlings infected with Ganoderma boninens and oil palm seedlings not infected with Ganoderma boninens. Table 1 also shows the results obtained by cutting the stem of the oil palm seedling vertically and calculating the ratio of the area of the lesion in the vertical cross-sectional area. [Table 1] As is apparent from Table 1, oil palm seedlings infected with Ganoderma boninens and Ganoderma
There is a significant difference in the amount of proline between oil palm seedlings not infected with Boninens. In particular, from Table 1, it can be seen that the presence or absence of Ganoderma boninens infection in oil palm seedlings in which the proportion of the lesion area in the vertical sectional area is 20% or less can be detected. In other words,
According to this method, even in the early stage of infection when no symptoms due to Ganoderma boninens infection are detected, Ganoderma boninens
The presence or absence of Boninens infection can be diagnosed reliably. Further, in the above-described embodiment, the ratio of the area of the lesion spot to the vertical cross-sectional area of the stem of the oil palm seedling is 2.
It can be seen that even in a very small case such as 8%, a significant difference is observed in the value of the proline amount. From this,
According to this method, the presence or absence of Ganoderma boninens infection can be reliably diagnosed even in the early stage of Ganoderma boninens infection. In the above-described embodiment, the present method was applied to oil palm seedlings positively infected with Ganoderma boninens to diagnose the presence or absence of infection. The oil palm which cannot be distinguished is diagnosed. That is, according to the present method, the oil palm of the diagnosis target is subjected to an acidic ninhydrin reaction between the oil palm of the diagnosis target whose presence or absence of the infection cannot be determined and the control oil palm of the uninfected control, and the results are compared. Ganoderma in
The presence or absence of Boninens infection can be diagnosed. As described in detail above, according to the method for diagnosing microbial infection according to the present invention, ganoderma is detected by detecting a predetermined amount of amino acids in an extract extracted from oil palm leaves. Diagnosis of infection of the genus Bacteria can be made. Therefore, according to the present method, the presence or absence of infection with Ganoderma spp. Can be diagnosed early and reliably without damaging the growth of oil palm.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a characteristic diagram showing a calibration curve showing the relationship between the absorbance of light having a wavelength of 515 nm and the amount of proline.

Continued on the front page (72) Inventor Marzuki Azahari Malaysia Selangor Daral Ahsan, Kajang 43000, Bandar Bar Bangi, Persian Run Institution, Number 6, Palm Oil Research Institute of Malaysia (72) Inventor Idris Abseman Selangor, Malaysia Darall Ahsan, Kajang 43000, Bandar Bal Bangui, Persialan Institution, Number 6, Palm Oil Research Institute of Malaysia (72) Inventor Mohammad Hanif Harun, Malaysia Selangor, Malaysia Daral Ahsan, Kajang 43000, Bandar Bar Bangi, Persian Run Institute, Number 6, Palm Oil Research Institute of Malaysia (72) Inventor Arifin, dulse, Malaysia Selangor Dararu Ehsan, Kajang 43000, Banda Lumpur Baru Bangi, Persian run Lee Nsutichushi, number 6, palm oil Research Institution capital of Malaysia (58) investigated the field (Int.Cl. ⁷ , DB name) C12Q 1/00-3/00 BIOSIS / WPI (DIALOG)

Claims (1)

(57) [Claims 1] An acid ninhydrin reaction is performed using oil palm leaves as a sample, and 515 n in the obtained reaction product is obtained.
A method for diagnosing microbial infection in oil palm, comprising detecting an infection of a genus Ganoderma by measuring the amount of an amino acid having an absorption maximum near m.