添加幾丁質對土壤性質、微生物菌相與植物生長影響之研究

Abstract

由於全球環保意識抬頭，各國政府陸續禁用高毒性化學農藥以及推廣降低化學肥料的使用量，對於環境友善的永續農法逐漸受到重視。有機廢棄物來源的土壤改良劑除了可以達成資源循環的目標，還可以改善土壤性質、促進植物生長或抑制植物病蟲害，成為施行永續農法時減少化學肥料以及化學農藥使用量的解方之一。其中，甲殼類廢棄物被認為因其主成分為幾丁質而具促進植物生長，甚至抑制植物病蟲害發生之功效，長久以來備受關注。然而，幾丁質是如何造成此效應之機制仍有許多未明之處。因此，本研究藉由將幾丁質添加到不同的土壤樣品，來探討幾丁質降解過程中，土壤物理化學性質之變化，並利用菌相分析各土壤於添加幾丁質後細菌族群組成和結構上的改變、細菌間的關聯性以及菌相可能的功能改變等，也實際種植小白菜來驗證幾丁質促進植物生長的能力。最後更基於菌相分析結果，從土壤中分離候選細菌菌株以驗證幾丁質是否可透過提高特定土壤微生物種類增強抑制植物病害的能力。本研究結果揭露三種測試土壤於添加幾丁質後會有不同程度但趨勢相似的物化性質改變，有效氮含量皆顯著提升，其中土樣 V 的有效氮含量隨時間上升的幅度最為明顯；有機碳含量和總氮含量在三種土壤樣本中的變化較不明顯，而三種土壤樣本的碳氮比與 pH 值皆隨時間下降。經由兩次孵育試驗的 16S rDNA 總體基因體分析後可得知，在施用幾丁質後的 15 天內，可降解幾丁質的細菌物種大幅度增加，到了後期，則出現較多可利用幾丁質分解後下游產物的細菌物種。進一步針對施用後初期的樣本進行共現網路及其關鍵物種的分析可以得知，添加幾丁質後細菌物種間的關係變得更為緊密；功能性基因體預測除了觀察到與微生物生長相關功能的基因豐度增加外，與幾丁質降解和硝化作用相關功能的基因豐度也均有上升的情形，凸顯幾丁質對於氮循環的貢獻。接著利用酵素活性分析驗證功能預測的結果，發現幾丁質酶與 N-乙醯葡萄糖胺酶的活性於添加幾丁質後在土樣 O 中增加最多。由於添加幾丁質後測到有效氮含量增加的現象，推測應可提高作物生物量，因此利用添加幾丁質後 90 天的土樣種植小白菜，結果發現無論是何種土樣，35 天大的小白菜生物量皆較未添加幾丁質的對照組高，其中土樣 C 的提升效果最為明顯，證實土壤中添加幾丁質可以促進植物生長，且可能是透過提高有效氮含量所致。最後根據生物資訊學分析結果，分離出添加幾丁質後在土壤中占比大幅提升的細菌屬菌株，包括 Chitinibacter spp. 和 Flavobacterium spp.，並以幾丁質培養基確認其可迅速降解幾丁質。對峙試驗中雖未觀察到其可抑制真菌菌絲生長的能力，但均有破壞南方根瘤線蟲蟲卵，並造成其孵化率下降的情形。綜言之，本研究透過使用更多土壤樣品的分析，了解幾丁質的添加可增加土壤中的有效氮含量，進而促進植物生長；雖然添加幾丁質造成微生物族群組成及結構的變化在各土樣中不盡相同，但均可觀察到參與幾丁質降解、功能相似的菌種數量或占比增加的情形，然而這些菌種是否是造成添加幾丁質能抑制病害效果的原因，仍有待後續研究探討。

As environmental awareness rises, authorities are gradually banning highly toxic chemical pesticides and promoting the lower usage of chemical fertilizers. Thus, environmentally friendly approaches to sustainable agriculture are considered important. Soil amendments derived from organic wastes are much more appealing as an alternative to chemical pesticides and fertilizers thanks to their effects on improving soil quality, promoting plant growth, and suppressing soil-borne diseases. Crustacean wastes are a rising star, given that the main component, chitin, is suggested to promote plant growth and suppress diseases. However, the mechanisms underlying these effects are still vague. Therefore, this study investigated how soil physicochemical properties were affected during chitin degradation after amending chitin into soil samples collected from different locations. Metagenomic approaches were then utilized to investigate how microbial communities derived from different soils respond to chitin amendment in terms of composition, structure, association, and function, and the plant growth-promoting effect of chitin was confirmed by planting Chinese cabbage in soil 90 days after chitin amendment. Finally, the bacteria were isolated from the representative soil samples based on bioinformatic information and further verified for their disease suppressiveness abilities. The data from this study revealed that in the presence of chitin, soil physicochemical properties changed differently in three tested soils with similar trends. The available nitrogen content changed the most, and soil sample V had the highest increase of available nitrogen content. There were marginal differences in soil organic carbon and total nitrogen, while soil pH and C/N ratio decreased for all three soil samples. Metagenomic analyses of two incubation experiments revealed that the chitinolytic taxa were greatly enriched during the early stage of chitin application (before 15 days), while the taxa utilizing downstream products from chitin degradation were enriched during the later stage of chitin application. Further co-occurrence analysis of the taxa from the early stage after the chitin amendment indicated that tighter associations among microorganisms in soil amended with chitin could be observed, and the number of keystone taxa also increased. Metagenome function prediction showed that in addition to the genes related to microbial growth, gene functions associated with chitin degrading and nitrification were significantly enriched, suggesting the contribution of chitin degradation to the nitrogen cycle. By determining enzyme activities to validate the results from functional analysis, it was shown that higher activities of chitinase and β-N-acetylglucoaminidase were detected in soil sample O upon chitin addition. The drastic increase in available nitrogen content implies the capability of enhancing crop biomass; thus, all soil samples collected after 90-day incubation with chitin were used for cultivating Chinese cabbage. The biomass of 35-day-old cabbages grown in the chitin-amended soil was higher, of which soil sample C had the most significant increase, supporting the hypothesis that enhanced available nitrogen content after chitin amendment could help promote plant growth. Finally, based on the metagenomic data, bacterial taxa enriched in chitin-amended soil were successfully isolated, including Chitinibacter spp. and Flavobacterium spp., and their chitinolytic activities were also confirmed using in vitro assays with chitin agar. Antagonistic assays revealed that these isolated bacterial strains had limited anti-fungal ability, but they can damage eggs of Meloidogyne incognita, leading to reduced hatchability. All in all, more soil samples were used to analyze the effect of chitin amendment in one study, and the results indicate that the available nitrogen content upon chitin amendment increased over time, regardless of the soil samples, contributing to enhancing crop biomass. Even though the soil microbial community composition shifted differently in response to chitin amendment in different soil samples, similar trends of increased number or proportion of chitinolytic bacteria could be observed. However, further research and investigations are required to truly understand whether those bacteria are the leading cause of suppressing soil-borne diseases.