Macro-microscopic evidence of pest, diseases and coexistence of microplastics in Avicennia marina leaves from Mangunharjo Village, Tugu District, Semarang, Central Java, Indonesia

There are enormous organisms that live depending on the mangrove ecosystem due to its important ecological services. However, their sustainability is threatened by the presence of diseases and pests. This study aimed to provide macro-microscopic evidence of diseases and pest infestation in Avicennia marina in Mangunharjo village, Tugu District, Semarang City, Central Java, Indonesia. During the survey, several trees had galls and symptoms of diseases on the leaves. Interestingly, we also found unidentified objects that were suggested as microplastic on the surface and inside of the leaves.


Introduction
Among all coastal plants, mangrove is noted as the most profiled plants including for its ecology, biodiversity, carbon stock and prospect as future biomedicine [1][2][3][4][5][6][7][8].Parida and Jha [9] stated that many mangrove species develop a special anatomic structure namely salt glands in their leaves to excreting the salt.Other species also able to conduct an ultra-filtration at the root cell membrane to exclude the salt, or accumulate the salt in their cells and tissue then prevent the damage by an efficient ion uptake into vacuoles in leaves, translocation to the outside of leaves, cuticle transpiration and efficient leaf turnover to salt release.This ability helps mangroves to survive in conditions of fluctuating salinity in tidal environments Indonesia possesses 43 species of true mangrove that consisted of several genes such as Aegiceras, Avicennia, Bruguiera, Ceriops, Excoecaria, Lumnitzera, Rhizophora, Sonneratia, Xylocarpus, etc [10].Moreover, plenty of other organisms are also depending their live to the mangrove ecosystem hence, this ecosystem is noted to be highly productive.It poses several ecological services that support other organisms to live, include human [11].Some mangrove ecosystems are located nearly to residency or urban are that also affecting the health condition of the mangrove [12].
Semarang is the capital city of Central Java Province which some areas are directly adjacent to the sea.Therefore, this condition supports several locations in Semarang to have mangrove forests, such as Mangunharjo beach in Tugu district [13].Avicennia alba, A. marina, Bruguiera sp., Rhizophora apiculata, R. mucronata and Sonneratia sp. have been reported previously from this location [4].In addition, this location is an urban mangrove forest that is near to residency.This condition allegedly leads to a high abundance of pollutant.Laila et al. [14] reported the presence of 3.585 particle microplastics per m 3 area in mangrove ecosystem around Mangunharjo village with fiber plastic as the most abundant.Moreover, Wulandari et al. [15] also reported the exposure of heavy metal to the mangrove plants around Mangunharjo village.Pollutant exposures to mangrove trees is noted to give negative impacts to mangrove health [16].
As mangrove health declines due to the pollution, pest and diseases in this ecosystem also threaten their sustainability [17].However, there is no study about pest and diseases in mangrove ecosystem from Mangunharjo village, yet.The purpose of this study was to assess the symptoms of pests and diseases that occur in mangrove leaves through microscopic observations.

Sampling and survey
Sampling was conducted in Mangunharjo village, Tugu District, Semarang City, Central Java (Figure 1).A purposive sampling was conducted by collecting the leaves that had symptoms of pest attacks and diseases infections.Sampling sites were adjacent to the street and close to the tourism sites.Samples were collected then kept inside zip lock plastics then transferred to laboratory.During the sampling, mangrove species and the pest's predation were recorded in-situ using iPhone 13 ProMax camera.

Microscopic observation
The fresh mangrove leaves were directly observed under the microscope (RELIFE RL-M2 Binoculer 7-45× magnification).The leaves with galls were dissected using cutter before observed under the microscope.Further, the symptoms of diseases and pest's infestation on mangrove leaves were captured using iPhone 13 ProMax camera.Then characterized using previous reports [17][18][19]

Result
In this location, several major mangroves were found such as Avicennia marina, Rhizophora apiculata and R. mucronata.This finding is suitable to previous reports that also only found these three species from the same location [20].During survey, gall was found in all A. marina trees in the sampling location, while leave diseases were found in site number 2 and 4 (Figure 2). Figure 2 shows the presence of leaf spot diseases were visible on the upper side which change the colour into yellow with irregular shape (A-B).On the other hand, fungal infection was spotted on the fruit that rotting the fruit and prevent the fruit development (C).Study related to mangrove-diseases in Indonesia is barely reported.Leave spot diseases could be induced by bacteria or fungi.However, it is lack of study about this disease in A. marina.Prior studies reported several fungal pathogens that infected other species of Avicennia spp.such as Alternaria alternata and Pseudocercospora avicenniicola [21,22].However, the symptoms that were caused by these two fungi are different with the symptoms that found in A. marina from Mangunharjo village, Semarang.Therefore, it is strongly suggested to perform a further study to isolate and identify the causative agent of this diseases.Then, the presence of pest infestation the leaves of A. marina is presented by Figure 3.The presence of eggs and larva of the unidentified insect inside of the galls in coherence with the previous studies.Several studies proved that the galls on the leaves of Avicennia were induced by insects such as Actilasioptera spp., Cecidomyia avicenniae, Meunieriella sp., etc. [24][25][26][27][28].Moreover, Kolesik and Gagné [28] stated that Actilasioptera falcaria is one of insect that causing gall on A. marina from Indonesia.In addition, genus Avicennia also well known as a "super host" due to the richness of gallinducing insects that live inside of its organ [24].
Bagworms have been noted as a pest in plantation industry.This insect is easily distinguished by the presence of "bag" structure that made of silk and dry plant's material, algae, lichen or dead insect to protect their body [29].The bagworms are mainly composed by Lepidoptera.Several of them are the larvae of moths that commonly reported as pest in mangrove such as Acanthopsyche sp., Cryptothelea sp., Hyblaea puera, and Pteroma plagiophleps [29][30][31].Overpopulation of bagworms causes bald leaves and triggers tree death.
Interestingly, during microscopic observation we found some unidentified objects that allegedly as microplastic.Figure 5 shows the appearance of the microplastic in and on the leaves of A. marina.The result of microscopic observation found that the unidentified objects were suggested as a microfiber with blue, white and black colours.We spotted some of the fibers were presence inside of the leaves.Microfiber is one of the types of microplastic that commonly found in the environment, include mangrove ecosystem.Microplastic is a micro-size plastic particle with 1 µm -5 mm.This pollutant is a big environmental problem for this past decade [32].Moreover, nanoplastic currently become an emerging environmental issue in the world.This pollutant has size < 1 µm which make the nanoplastic become easier distributed and transported from environment into the organism body [33] The presence of microplastic in mangrove ecosystem is not a new finding since plenty of studies reported this issue [34,35].However, most of the studies reported the presence of microplastic in the mangrove sediment.On the other hand, in this study we found the microplastic on the surface and inside of the leaves.
The presence of microfiber on the surface of the leaves is suggested due to the influence of the wind movement that brings the pollutant.Since the sampling location is adjacent to the street and tourism site with high plastic pollution.Nevertheless, the mechanism of how microfibers can be present inside of leaf organs is still unknown.In addition, a new hypothesis was occurred due to this phenomenon about the contribution of the pests to the existence of fibers inside of the plant tissue.So far, the similar study has been conducted on nanoplastic.It is proven that the naturally occurring of transpiration pull in plants aids the translocation of nanoplastics within plants [33,36].Besides, the existence of microplastic and nanoplastic is noted to give negative effect to the plant physiology and biochemical such as late germination, delay the root growth, triggered oxidative stress and induced cyto/genotoxicity on root system [36][37][38].

Conclusion
Insect and bagworms infestation on the leave of A. marina were found in Mangunharjo village.Microscopic observation successfully showed that the occurrence of leaf gall was induced by the insect.Moreover, the diseases that found in the location infected leaves and fruits.In addition, this study also found the presence of microfiber on the surface and inside of the leaves.Due to the interesting finding in this study, we suggested a further investigation using DNA barcoding to identify the pest and diseases agents.In addition, this study triggers a new research question about the pests contribution to the existence of fibers inside of the plant tissue.

Figure 4 .
Figure 4.AM1-AM3 show the formation of gall on the leave of A. marina.Santos et al.[23] stated that the formation of gall is an abnormal growth of cells, tissue, or organ in plants that is induced by the invasion of insects, fungi, and nematodes.A microscopic observation was conducted in this study to

Figure 4 .
Figure 4.The presence of eggs (AM7) and larva (AM8-AM9) in the gall on the leaves of A. marina.

Figure 5 .
Figure 5. Unidentified objects that were suspected as microplastic that were exist inside and on the surface of A. marina leaves.(A, D, F) on the surface, (B, C, E) on the surface and inside of a leaf.