Long-term fungal inoculation of Ficus sycomorus and Tectona grandis woods with Aspergillus flavus and Penicillium chrysogenum

In the current study, two molds, Aspergillus flavus (ACC# LC325160) and Penicillium chrysogenum (ACC# LC325162) were inoculated into two types of wood to be examined using scanning electron microscopy-energy dispersive X-ray (SEM–EDX) and computerized tomography (CT) scanning. Ficus sycomorus, a non-durable wood, and Tectona grandis, a durable wood, were the two wood blocks chosen, and they were inoculated with the two molds and incubated for 36 months at an ambient temperature of 27 ± 2 °C and 70 ± 5% relative humidity (RH). The surface and a 5-mm depth of inoculated wood blocks were histologically evaluated using SEM and CT images. The results showed that A. flavus and P. chrysogenum grew enormously on and inside of F. sycomorus wood blocks, but T. grandis wood displayed resistance to mold growth. The atomic percentages of C declined from 61.69% (control) to 59.33% in F. sycomorus wood samples inoculated with A. flavus while O increased from 37.81 to 39.59%. P. chrysogenum caused the C and O atomic percentages in F. sycomorus wood to drop to 58.43%, and 26.34%, respectively. C with atomic percentages in Teak wood’s C content fell from 70.85 to 54.16%, and 40.89%, after being inoculated with A. flavus and P. chrysogenum. The O atomic percentage rose from 28.78 to 45.19% and 52.43%, when inoculated with A. flavus and P. chrysogenum, respectively. Depending on how durable each wood was, The examined fungi were able to attack the two distinct types of wood in various deterioration patterns. T. grandis wood overtaken by the two molds under study appears to be a useful material for a variety of uses.


Inoculation of wood samples with the tested fungi.
Each piece of wood sample was individually inoculated in Petri plates with media of potato dextrose agar (PDA) and fungus discs from A. flavus (ACC# LC325160), and P. chrysogenum (ACC# LC325162) measuring 5 mm in diameter 9,36 . Following this, fungi were cultured on wood blocks under experimental conditions (27 ± 2 °C and 70 ± 5% relative humidity (RH)), where the incubation lasted until the complete colonization of the fungus on the Petri dishes and wood block. After this time, Petri plates were covered a plastic wrap made of cling film and stored in a growth chamber for 36 months at the ambient conditions (27 ± 2 °C and 70 ± 5% RH) 28 . The inoculated woodblocks were then removed for additional examination.

SEM-EDX examination of the inoculated wood samples.
Using an environmental scanning electron microscope (ESEM) (Quanta FEG250; FEI Co., Hillsboro, OR, USA), the inoculated wood samples of F. sycomorus and T. grandis were analyzed for the fungal infestation (A. flavus and P. chrysogenum) on the surface and at 5-mm depth of the infected wood block. Energy dispersive spectroscopy (ESEM-EDS (Quanta FEG250; FEI Co., Hillsboro, OR, USA, with tungsten electron source, at 20 kV) was used to examine the variations in surface elemental compositions.
CT scanning examination. The hyphal fungal infestation within the tested wood samples was examined by X-ray computed tomography (CT) scanning using a Toshiba Aquilion 16 CT Scanner, Tokyo, Japan. Using 3-D imaging, which is an image quality with surface shaded-renderings and volume-rendered 3-D images, datasets were displayed and images and videos were recorded. Distance measurements were made as the 3-D surface was zoomed in and out. The Aquilion 16 features 896 channels in 40 rows of solid-state detectors with a variety of slice thicknesses was used to test high image quality. In the x, y, and z directions, the system had a low-contrast resolution of 2 mm at 0.3% and a high-contrast resolution of 0.35 mm. The following values were used: voltage 120 kV, current 150 mA, timing 15.819 s, and thickness 0.5 × 16 mm 237 . The CT number, which is expressed by brightness data in an image, is based on linear X-ray absorption coefficients 38 .

SEM and CT scanning examination results of fungal inoculated Ficus sycomorus wood. On
Ficus sycomorus wood, Aspergillus flavus growth was clearly visible in all samples (Fig. 1). A visual augmentation in conidia quantity was observed at different areas with Several aerial fungal mycelium and conidia covered the surface of F. sycomorus in various locations, causing a visible increase in conidia abundance ( Fig. 1a and b). Additionally, conidiospores and mycelium quantity increased as a result of aberrant spore development along the hyphae in the basal mycelium ( Fig. 1c and  www.nature.com/scientificreports/ The SEM images of the analyzed F. sycomorus wood sample that was 5 mm deep and infected with A. flavus are displayed in Fig. 2a and b. The infected wood had A. flavus at its ideal density, which resulted in a notable increase in hyphal development and spore generation. After 36 months of incubation, A. flavus was detected inside the wood by CT scanning, as shown in Fig. 3.
The production of A. flavus conidial, however, may rise even below the surface, showing that A. flavus can rot F. sycomorus wood through the pits and within cell walls. Additionally, among the wood fibers, the spore and hyphae structure was visible. In a previous study, it was discovered that various molds, including Botryodiplodia theobromae, Trichoderma longibrachiatum, A. candidus, A. ustus, and A. terreus quickly degraded F. sycomorus wood 39 . In addition to the great porosity of the wood, which allowed these aerobic fungi to grow through it, the low level of antimicrobial chemicals in F. sycomorus may have contributed to the enormous development of A. flavus 39 . Figure 4 depicts the SEM morphology of P. chrysogenum colonies on wood after 36 months at 27 ± 2 °C. It was possible to see P. chrysogenum, which had peridial hyphae with thick-walled dichotomously branching walls and a short, bifurcating appendage resembling a spine. The center displays a gymnothecium of the globose open reticulum type with ascospore mass. Ascospores and developing asci are seen. It is common to find tiny, radially projecting peridial appendages that resemble deer antlers.
In the wood of F. sycomorus, P. chrysogenum produced hair baits (Fig. 4a). The ascomata appeared to have septate, branching, and thick-walled peridial hyphae, but lacked the distinctive boathook-shaped appendages. Globose-to subglobose asci were present. Under SEM, the spores were seen to be irregular and rough ( Fig. 4b through d).
In addition to severely degraded and weakened cell walls, SEM images of F. sycomorus wood degradation after 36 months also revealed colonized hyphae in cell walls. The total disintegration of the cell wall and vessels may be linked to the severe chemical materials degradation of the cell wall [40][41][42][43] . As previously indicated, After 3 months of incubation with A. niger, identifiable notches of cell wall erosion and cavities created by fungal hyphae within the cell walls were discovered in wood., while P. chrysogenum only created erosion troughs formed in cell walls while colonizing F. sycomorus wood 5 .
SEM images of a 5-mm-deep incision in the infected wood were taken to demonstrate the extent of P. chrysogenum inside F. sycomorus wood ( Fig. 5a through d). Images revealed P. chrysogenum inside wood had grown enormously. After 36 months of incubation, a CT scanning (Fig. 6) of the wood demonstrated A. flavus penetration growth.
The SEM images of wood samples from F. sycomorus showed that the cell walls were distorted, had some structures holes, and were missing entire wood cells. These are the results of the development of fungus hyphae and spores 29 . Cell wall layers have likely detached and separated as a result of soft rot fungal growth 20,44 . Despite poor structural preservation, a fungal microbial degradation was discovered in archeological F. sycomorus wood 2 . After 4 months of incubation with Penicillium chrysogenum, the secondary wall layers of F. sycomorus wood were disrupted as a result of severe cell wall breakdown 5 .

SEM and CT scanning examination of incubated Tectona grandis wood.
In all teak wood samples, the growth of A. flavus was minimized ( Fig. 7a and b). The spore loss is visible in the SEM images. The branching and decay are the two main modifications to the hyphal morphology. According to Fig. 7c and d, phenolic and After 36 months on incubation, teak wood infected with Penicillium chrysogenum showed reduced mycelial growth ( Fig. 9a through d). These findings suggested that the conidial production of P. chrysogenum might not grow in teak wood due the presence of phenolic and other aromatic antimicrobial compounds, such as anthraquinines and tectoquinones [45][46][47][48][49][50] . The chemical compounds in the wood changed the morphology of P. chrysogenum, colony morphology, and multicellular clumps that lost the spore.
It was discovered that the size of the mycelial pellets had significantly decreased, the cell wall had absorbed, and the shape of the mycelial pellets had changed. Nearly no growth indicators of P. chrysogenum were visible on the surface and core of the wood during the CT scanning (Fig. 10).

EDX measurements
Elemental composition changes of Ficus sycomorus wood. Incubated F. sycomorus wood with A. flavus and P. chrysogenum for 36 months is shown in Fig. 11 to have different elemental compositions from uninoculated wood. In the un-inoculated wood ( Fig. 11a and Table 1), The atomic percentages of C and O in the un-inoculated wood were 61.69%, and 37.81%, respectively, while they changed to 59.33% for C and 39.59% for O in the inoculated wood samples with A. flavus (Fig. 11b and Table 2). The C and O atomic percentages in wood samples inoculated with P. chrysogenum for 36 months dropped to 58.43%, and 26.34%, respectively ( Fig. 11c and Table 3).
The un-inoculated wood contained 0.10% atomic percentage of K. In the infected wood with A. flavus, and P. chrysogenum, this climbed to 0.34% and 2.09%, respectively. Additionally, the Ca content rose from 0.18% in the control wood sample to 0.35% with A. flavus infected wood sample and to 1.81% in wood sample inoculated with P. chrysogenum.  www.nature.com/scientificreports/ Elemental composition changes of teak wood. As compared to the un-inoculated sample, Fig. 12 illustrates the changes in the elemental composition of teak wood that was incubated for 36 months with A. flavus and P. chrysogenum. In the un-inoculated wood sample, C and O are the two most prevalent elements with atomic percentages of 70.85% and 28.78%, respectively ( Fig. 12a and Table 4). As teak wood was inoculated with A. flavus (Fig. 12b and Table 5) and P. chrysogenum (Fig. 12c and Table 6) for 36 months, the atomic percent- Figure 7. SEM images of teak wood that was surface-exposed to A. flavus for 36 months (a,b) and after cutting of 5-mm from infected wood (c,d). IH internal hyphae, SP sporangiophore, C conidium: FCW fiber cell wall, VCW vessel cell wall, BP bordered pits.   www.nature.com/scientificreports/ study. Molds are typically discovered and grow in moisture-damaged wood 51 , create colored spores and large amounts of pigment on the surfaces of wood, which decrease the quality of the wood 52,53 , but do not influence the strength of the wood 54 .
The carbon-rich components of wood are reported to be metabolized by molds and fungi that disintegrate wood. This produces massive fruiting structures of fungi, which release a huge amount of spores into the natural environment 10,55 . Molds that are unable to depolymerize the primary chemical polymers of wood (cellulose, lignin and hemicelluloses) can consume the sugars and starches found in ray and axial parenchyma cells lumen 56 . Through pores and pits, The hyphae of the fungus can enter the cell walls through holes and crevices 57 .
The two mold fungi-inoculated woods showed that the C element content was lower than it was in the control sample. This outcome is consistent with previous work 10 . Additionally, it was found that the molds absorbed the C sources 58 . When growing on Fagus sylvatica wood, molds like P. selerotigenum and A. niger consume a lot of C, but P. selerotigenum consumes a lot of C content when growing on Juglans nigra wood. In contrast, minimal  According to the tests of adhesion in the study of Soumya et al. 59 , P. chrysogenum was unable to adhere to the cedar wood substrate, although P. granulatum, P. crustosum, and P. commune were able to do so, contrary to what was theoretically expected. The development of P. chrysogenum PCL501 on wood waste results in the production of the xylanase enzyme, which is most strongly induced by the carbon source 60 . When cultured on a www.nature.com/scientificreports/ bran-wood flour-olive oil or a bran-soy bean media, the water-soluble enzyme (Lipase) generated by P. oxalicum and A. flavus was capable of hydrolyzing the olive oil 61 . The lignin structure in agricultural lignocellulosic wastes was discovered to be degraded by the strain of A. flavus EGYPTA5, which secretes lignin peroxidases, nitrate reductase, laccase, polyphenol oxidase, and cellulase enzymes, without changing the concentration of cellulose 62 . It was discovered that several A. flavus fungal  www.nature.com/scientificreports/ isolates produced cellulase-free xylanase in a variety of soil environments, including manures, dead and decaying wood, and soil samples 63 . A. flavus produced the most cellulase enzyme when it was cultivated on wood sawdust, according to study 64 . A. flavus was isolated from its natural environment including wastewater, rotting wood, wheat straw, and field soil samples, and it produced laccase enzyme 65 . Both SEM and CT scanning examinations confirmed the growth of molds on the studied wood samples on the surface and core showing the structural growth of fungi.

Conclusions
The dispersion of the two molds' fruiting structures, spores, and hyphae that covered the damaged wood surfaces after 36 months of incubation could be clearly seen by the examination instruments SEM-EDX and CT scanning. According to the study, the carbon-rich components of the examined Ficus sycomorus and Tectona grandis woods are metabolized proportionately by Aspergillus flavus and Penicillium chrysogenum. The findings supported the long-term durability and the non-durability phenomena of Tectona grandis and Ficus sycomorus woods, respectively. Finally, the surface and core of the analyzed wood samples showed structural growth of fungi, which was validated by SEM and CT scanning studies.