Jing Li · Yan Yu · Chao Feng · Hankun Wang

Abstract Characterizing the mechanical properties of wood cell walls will lead to better understanding and optimization of modifications made to wood infected by the blue-stain fungi. In this study, in situ nanoindentation was used to characterize the mechanical properties of the cell walls of Pinus massoniana infected by blue-stain fungi at the cellular level. The results show that in situ nanoindentation is an effective method for this purpose and that blue-stain fungi penetrate wood structures and degrade wood cell walls, significantly reducing the mechanical properties of the cell walls. The method can also be used to evaluate and improve the properties of other wood species infected by blue-stain fungi.

Keywords Blue-stain · Nanoindentation · Cell wall · Pinus massoniana · Mechanical properties

Introduction

Masson pine (Pinus massoniana Lamb) is one of the most economically significant and widely used fast-growing tree species in China (Bardage et al. 2014), but fungal attacks can result in heavy economic losses (Zink and Fengel 1990). Blue-stained wood refers to wood that is infected by blue-stain fungi, which feed on simple sugars and starches in the wood and consequently cause bluish or greyish discolorations on its surface. Blue-stain fungi are a common type of fungus that only infects the sapwood. Masson pine infected with blue-stain fungi are more susceptible to attacks by decomposing fungi, leading to a shortened product life (Su et al. 2005; Zhao et al. 2005). Over the decades, numerous studies have been devoted to bluestained wood, with the majority focusing on blue-stain fungi’s infection mechanism (R?tt? et al. 2001; Robinson et al. 2013, 2014a, b), microstructure and performance(Thaler et al. 2012), and the technology to protect and modify against infection. These studies have assessed treatment methods that alleviate the adverse effects of bluestain fungi on the surface of infected wood, such as chemical biocides (Kingsbury et al. 2012; Liu et al.2012, 2014), natural products and derivatives (Yang and Clausen 2007; Hsu et al. 2009; Shreaz et al. 2011; Liang et al. 2013; Bardage et al. 2014), compression and thermal modification (Yu et al. 2011a; Mazala et al. 2004; Kocaefe et al. 2008), plasma treatment and bleaching (Evans et al.2007; Jamali and Evans 2013), and fungal pigments(Robinson et al. 2014a, b; Hernandez et al. 2016). These methods also help improve certain mechanical and functional properties of the wood.

Currently, studies on the impact of blue-stain fungi on the mechanical properties of wood are inconclusive. Some researchers believe that blue-stain fungi merely occur on the surface, which decreases the aesthetic value of the wood, but seldom affect its mechanical properties. Because blue-stain fungi only feed on substances with low molecular weight in the wood, they do not degrade wood cell walls (Schmidt 2006; R?tt? et al. 2001). Some researchers argue that although blue-stain fungi’s penetration to the wood structure via medullary rays and colonizing tracheids can increase its permeability, it will not have a significant influence on the mechanical properties or result in decay.The reason is that increased permeability is believed to be induced by a selective degradation of pit membranes in the bordered and half bordered pits, resulting only in trivial changes in the tracheid cell walls (Greaves 1970; Seifert 1993; Mai and Militz 2004; Knaebe 2002). However, these studies only measured certain mechanical properties such as compression parallel to the grain, bending strength,toughness and hardness, and none of the results suggest that a significant decrease in strength was caused by bluestain fungi (with the exception of a marginal increase to wood permeability and loss of flexibility) (Findlay 1959;Wingfield et al. 1993).

Still, other studies have demonstrated that blue-stain fungi can indeed result in the alteration of mechanical properties of wood (Schmidt 2006). These studies showed that blue-stain fungi could penetrate and decompose internal wood structures, leading to a decrease in dry mass,toughness, surface hardness and flexibility (Chapman and Scheffer 1940).

Mechanical tests at the macroscopic level are still the primary means to assess the extent of blue-stain fungi’s impact and the effectiveness of modification methods, with no precise characterization at the cellular level. It is well documented that at the microscopic level, mechanical behavior deviates significantly from the classic elasticplastic model because of the so-called size effect (Zhu et al.2008). That means the characterization of blue-stain fungi mechanism at the macroscopic level is hardly comprehensive. Therefore, it is necessary to conduct in situ monitoring of mechanical property changes of blue-stained wood at the cell wall level.

Nanoindentation is a powerful method to test hardness and the elastic modulus of materials at the micrometer level (Wimmer et al. 1997). It has been increasingly used to mechanically characterize plant tracheids. In this study,blue-stained Masson pine was subjected to in situ nanoindentation to investigate changes in mechanical properties of infected cell walls. Samples were not embedded in any material to exclude the effects of resin and maximize the accuracy of the mechanical test. The technology used in this study can also be used to improve methods to enhance the properties of blue-stained wood and analyze the modification mechanisms of blue-stain fungi.

Materials and methods

Sample preparation

Appropriate sample preparation for the nanoindentation technique is crucial. Mature blue-stained sapwood of Masson pine (Pinus massoniana Lamb, approximately 30 years old was taken from a plantation in Huangshan,Anhui Province, China (Fig. 1a) was selected and cut into 5 mm × 5 mm × 20 mmcubes(radial × tangential × longitudinal). To prepare the surface, a four-sided pyramid (Fig. 1c) was cut with a sliding microtome (Leica SM2000, Germany). Then, the tip of each pyramid was further shaved with a glass knife, then a diamond knife,leaving a tiny, smooth surface on the top (Fig. 1d).

In situ nanoindentation testing and imaging

Nanoindentation was performed using a triboindenter(Hysitron, Minneapolis, MN, USA) with a Berkovich diamond tip (tip diameter of the tip ≤100 nm). Samples were tested in a hermetic chamber at 23 °C and 40% relative humidity. Different regions for each sample (Fig. 2a) were selected for indentation. Surface images were scanned before and after the indentation process to select valid indents for data analysis (Fig. 2b-d). The tests were performed in a force-controlled mode. With a maximum force of 250 μN(yùn) and a loading rate of 50 μN(yùn) s-1, 6 s constant loading was followed by 3 s of unloading. The elastic modulus and hardness of samples were then measured using the approach described by Cave (1978) and Yu et al.(2011b).

Results and discussion

As shown in Fig. 2, tracheids with different degrees of blue-stain invasion can be easily categorized into three groups, namely ‘‘normal’’, ‘‘mild’’, and ‘‘severe’’, divided by the surface roughness and the distance to the central blue-stained regions. The ‘‘normal’’ group was the control sample, which was chosen near the blue-stained area instead of other normal areas to avoid differences caused by other factors such as the microfibril angle (MFA), which may also influence the mechanical properties (Wagner et al. 2014; Yu et al. 2014). The surface of normal tracheid cell walls (Fig. 2b, uninfected cells) was smooth in comparison to the infected cell walls (Fig. 2c, d), where signs of shrinkage and numerous microcracks were clearly visible. These symptoms were caused by the degradation of saccharides in the cell wall where blue-stain fungi were found, which may partially account for the increase in permeability in infected wood as previously reported(Schmidt 2006; Robinson et al. 2013). For this reason,samples were not during sample preparation. For normal tracheids, epoxy resin infiltration into the cell lumina may also affect results. Although Kim et al. (2012) found that epoxy resin cannot penetrate the dense cell walls, Meng et al. (2013) reported significantly higher values for embedded wood cell walls. For the infected cells, epoxy resin will fill the cracks of the cell walls and enhance the mechanical properties of the wood.

Fig. 1 Flow diagram showing the process of preparing blue-stainedformed on the top of each sample d final surface formed on the top of Masson pine samples: a blue-stained Masson pine, b samples cut intosamples where indentation took place cubes of 5 mm × 5 mm × 20 mm (R × T × L), c pyramid shape

Fig. 2 Images of in situ nano-indentation on blue-stained Masson pine: a the region selected for analysis; b-d high magnification images of the same area obtained from indenter tips after indentation.The scanning size was 20 μm × 20 μm

Fig. 3 Box-and-whisker plots of a elastic modulus and b hardness of blue-stained Masson pine against increasing severity of decay as measured by nano-indentation

Figure 3 presents the box-and-whisker plots of the elastic modulus and hardness of tracheids cell walls in the three regions. Consistently, the presence of blue-stain fungi degraded the mechanical properties of wood due to the loosening of the infected cell walls. To a considerable extent, this loosening is apparently due to the extension of blue-stain fungi beyond the wood rays (where infection is typical) and their direct penetration of the tracheid walls.The average elastic modulus value of the normal samples was 19.3 GPa, similar to the experimental result of the same species under the same moisture content (MC)reported in a previous study (Yu et al. 2011c).

Hardness, determined by lignin content in the tracheid cell wall (Gindl et al. 2002) is another important indicator of cell wall strength, which helps material to resist permanent plastic deformation. By comparing the two sets of data, a difference of nearly 80 MPa (≈14.5%) was found,possibly caused by different levels of lignin content in the cell walls as a result of various growth environments. Gindl et al. (2002) found that the cell wall hardness of Norway spruce increases significantly with lignification and stabilizes when the lignification is complete. This finding is helpful for explaining the results of this study.

A qualitative description of decay severity is plotted against the elastic modulus of cell walls in Fig. 3a. As the severity of decay increased from ‘‘normal’’ to ‘‘severe’’, the elastic modulus dropped from 19.3 to 14.2 GPa, a 26.4%decrease. This reduction was close to the figures reported by Chapman and Scheffer (1940), where the toughness of infected pinewood generally dropped by 15-30%. This result supports the hypothesis that blue-stain fungi are responsible for the decrease in toughness in masson pine.Similarly, Fig. 3b shows the negative relationship between wood cell hardness and the severity of decay, with an overall drop in hardness of 12.5%. Matti (1938) and Mayer-wegelin et al. (1931) suggest that the surface hardness of blue-stained wood may be down by 10% at most. Therefore, the hardness of cell walls can also reflect the change in surface hardness (though not perfectly).Figures 3a, b also show that the impact of blue-stain fungi on hardness is lower than that on elastic modulus. This finding may be explained by the biological characteristics of blue-stain fungi, which extract hemicellulose (as opposed to lignin, which is responsible for cell wall hardness) from the polysaccharide chitin chains of wood cell walls to support the growth of melanized hyphae.

It is obvious that the experimental results of this study prove that blue-stain fungi do indeed reduce the mechanical properties of wood cell walls. However, it is uncertain whether the sample was only eroded by blue-stain fungi. In fact, the conditions that favored the development of bluestain fungi also stimulated the development of decay fungi.Therefore, decay fungi and blue-stain fungi may both be responsible for the wood degradation, in accord with many studies (Daniel 2003; Behrendt et al. 1995; Eriksson et al.1990). Hence, it is often good practice to examine bluestained wood for signs of decay (Chapman and Scheffer 1940). Meanwhile, some species of blue-stain fungi are pathogenic and are carried by aggressive bark beetles,which can cause severe damage to living trees (Molnar 1965; Horntvedt et al. 1983; Redfem et al. 1987; Peng et al.1996). The bark beetles thus act as vectors for the bluestain fungi. Solheim argued that the blue color commonly found in beetle-infested mountain pines is caused by several species of blue-stain fungi, which are vectored into the host trees by the beetles’ mycangia (Solheim 1995).

Based on the above analysis, it is possible to infer that the initial presence of blue-stain fungi has little effect on the mechanical properties of wood, and the impact of the fungi is minimal because of the limited extent of hyphae and damage to wood cell walls. However, the presence of blue-stain fungi does lead to the loosening of cell wall structures of wood. The mechanical properties of wood would eventually decrease if other agents such as decay fungi and beetles intrude into wood cell walls in the bluestained areas of wood. Evidently, further analysis is needed on the correlation between different types of blue-stain fungi, morphology and distribution of mycelium and the changes in blue-stained wood microstructures.

Conclusion

In situ nanoindentation is an effective method to study the mechanical properties of blue-stained masson pine. Bluestain fungi penetrate wood structures and degrade wood cell walls, and the nanoindentation tests indicate that bluestain fungi could decrease the elastic modulus by 26.4%and hardness by 12.5%. However, this may be due to synergistic effects caused by blue-stain fungi and decay fungi. The method used in this study can also help to enhance the properties of wood infected by blue-stain fungi.