Towards a laser-based irradiation system for attacking Curvularia sp.: Preliminary results

In the seedling stage, oil palm plants are susceptible to several diseases. Leaf spot disease caused by fungi or bacteria often appears that can lead to a failure and even death of the seeds. Spraying fungicides is usually conducted to diminish the fungi growth on the leaf. However, a continuous application of fungicides can become a resistance condition of the fungi to the given fungicides. In this research, a laser-based irradiation system is proposed to attack the fungi growth on the leaf. Photon as an electromagnetic wave is expected to penetrate the fungal cell, yielding dormant DNA, subsequently leading to the fungi’s death. The photon energy’s suitability could influence this technique’s effectiveness according to the absorption energy of the targeted fungal/bacterial cell. This work presents a preliminary result on the generated light power and the beam area at ∼16.59 mW/cm2 and ∼24 cm2, respectively, on Curvularia sp. under laboratory conditions.


Introduction
Plant nurseries are essential in plantations or cultivation since they determine production results.In the context of oil palm nurseries, a leaf spot disease commonly attacks the oil palm seedlings, mainly caused by fungal pathogens, such as Curvularia eragrostidis, Curvularia sp., Drechslera halodes, Cochliobolus carbonus, Cochliobolus sp., and Pestalotiopsis sp [1].Among these pathogens, Curvularia sp. is most frequently appears during the nursery period in oil palm [2].When the spot coverage rate exceeds 90%, curvularia leaf spot disease can cause death in oil palm seedlings [3].Therefore, it needs to be accompanied by appropriate control measures.In the process, the fungal spores spread through wind or water.If the oil palm seedlings are exposed to leaf spot disease, physical signs of small round spots (translucent yellow) appear, as depicted in Figure 1.Fungicides are generally applied to handle leaf spot diseases [4].However, frequent application may lead the pathogen to resist the fungicides [5] and could also be lethal to humans, animals, and plants [6].Therefore, this study is aimed to assess the inhibitory effect of several medium-power lasers with different wavelengths and energies on the growth of Curvularia sp. in vitro.The approach proposed in this study will be a more environmentally friendly option for eradicating leaf spot disease in oil palm nurseries.

Method
We built an irradiation house for laboratory applications, as shown in Figure 2. The architecture is a pyramid shape with plastic optical fiber access arranged on its top.The optical fiber is sourced from a laser launched at the first end of the optical fiber.Since the laser light was coupled through a lens interface, we employed a plastic optical fiber with a diameter of 400 µm to propagate the beam mode through the fiber inside efficiently.Here, the focus selection of the lens and its adjustment angle determine the light coupling's effectiveness [7,8].On the other side, the fiber's second end with a numerical aperture (NA) of 0.25 is left free, in which the laser is spout forming a light beam spot.The laser beam spot is further characterized to obtain a profile of power distribution per cm 2 , which is conducted by adjusting the distance between the light aperture and the target sample.The space is tuned every 1 cm resulting in a circle-shaped beam spot where the diameter and the light power can be measured.This measure allows having a power variation of the beam due to light divergence that is subsequently examined on the target sample.Fiber aperture Laser source system prepared as the irradiation sources, i.e., blue (λ = 405 nm), red lasers (λ = 638 nm), and infrared (λ = 808 nm) with the coupled power of ~400 mW.All the laser sources are linked to the optical fiber and radiate in the irradiation house.A control sample without irradiation exposure was included.Fungal colonies were further examined in fungal growth areas using Image-J software.We expect that a particular parameter of laser light could work propitiously to attack and resist the fungal cell life.Broadening retardation is taken into account as a fruitfulness indication.

Result and Discussion
This work employed an infrared laser with an excitation wavelength (λ) of 808 nm for investigating the optimized beam spot.The power at the optical fiber aperture was measured at ~400 mW, which was subsequently observed for a 1 cm increment until the beam spot area was similar to a petri dish (diameter, D = 9 cm). Figure 4 presents the power distribution per cm 2 due to distance (space) adjustment between the light aperture and a planar plane.From the laser aperture, the beam propagates outward and slowly diverges with distance, showing a broader beam spot and exponentially decreased light power.For fungal cell irradiation, a space between the fiber aperture and the targeted fungi sample was set at 12.5 cm, which resulting a beam power and spot area of ~16.59 mW and ~24 cm 2 , respectively.The selected area was appraised sufficiently to irradiate the targeted sample since the average maximal size occurred under ~20 cm 2 after three days.The fungi area growth was inspected for three days of investigation.As illustrated in Figure 5, after 7 hours of irradiation, the fungi area was measured the following day.Figure 6 summarizes the percentage of the growth increment compared with the original size on the initial day.There were three groups of irradiation samples, each consisting of three fungal samples in the petri dish.These results show that laser irradiation can impede fungal growth, whereas infrared irradiation presents the most effective.The size of fungal colonies increased at ~56.8% after infrared irradiation on the 3 rd day.It indicates the slowest growth compared to the other irradiation samples, which showed area increments of 88.3%, 70.4%, and 103.4% for the blue, red, and reference samples (control).Table 1 summarizes in detail the growth performances of laser irradiation that showed blue, red, and infrared laser irradiation can inhibit the growth of Curvularia with inhibition rates of 14.6%, 31.8%, and 45%, respectively.Here, the infrared laser exhibited a better inhibitory effect than the other sources.This irradiation technique presents a prospective result compared to a similar study on LED irradiation systems for attacking Botrytis cinerea [9].The latter presented purple and blue LEDs irradiation on tomato leaves for 12 h can penetrate the Botrytis cinerea growth with 22.3 and 15.16% inhibition rates, respectively.Other research demonstrated gamma irradiation showed a 90% inhibition rate of spore germination and mycelial growth of Botrytis cinerea at 4.0 kGy in vitro [10].However, it must be noted that gamma rays are a radiation hazard for the human body, which needs special protection infrastructures.

Conclusion
A physical approach to address the problem of fungal pests in production plants needs to be continuously developed.Indeed, the application of fungicides can rapidly eradicate fungal pests, but long-term fungicides will cause the mutation of fungal cells, resulting in fungicide resistance.In addition, fungicides could also be susceptible to contaminating the soil and environment.In this work, the application of the irradiation laser exhibits a promising result for effectively controlling fungal pathogens such as Curvularia sp.In this work, the infrared irradiation laser could retard the growth of Curvularia sp. at ~56.8% and an inhibition rate of 45%.Further research is needed to investigate a better irradiation parameter, efficient and practical irradiation time, and ensure the fungi perished.

Figure 1 .
Figure 1.Oil palm leaf is infected by fungal pathogens, primarily due to Curvularia sp.

Figure 2 .
Figure 2. Experiment setup of laser irradiation system for fungal pathogen attack.Furthermore, the second inspection is to observe the response of the Curvularia cells (Figure 3(a)) as the irradiation-target-sample placed on a transparent closed petri dish (Figure 3(b)).Three lasers were

Figure 4 .
Figure 4.The pattern of laser power distribution shows a decreasing trend of laser power with aperture-sample distances.On the contrary, the beam spot area is getting wider.

Figure 5 .
Figure 5. Illustration of sample growing in four days observation.

Figure 6 .
Figure 6.Percentage of area increment of Curvularia under laser irradiation system.

Table 1 .
Curvularia's growth increment under the laser irradiation and control samples (unexposed)