WO2021073033A1 - 一种降低生态繁育冬虫夏草砷及重金属含量的方法 - Google Patents

一种降低生态繁育冬虫夏草砷及重金属含量的方法 Download PDF

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WO2021073033A1
WO2021073033A1 PCT/CN2020/079156 CN2020079156W WO2021073033A1 WO 2021073033 A1 WO2021073033 A1 WO 2021073033A1 CN 2020079156 W CN2020079156 W CN 2020079156W WO 2021073033 A1 WO2021073033 A1 WO 2021073033A1
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humus soil
soil
humus
morel
bag
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French (fr)
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李文佳
李全平
蒋成吉
吕延华
张宗耀
邱健健
李明星
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东莞市东阳光冬虫夏草研发有限公司
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Publication of WO2021073033A1 publication Critical patent/WO2021073033A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/20—Culture media, e.g. compost
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/40—Cultivation of spawn

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  • the invention relates to the technical field of breeding high-quality cordyceps by reducing soil pollution through microbial restoration, and specifically relates to a method for reducing arsenic and heavy metal exceeding standards in ecologically breeding Cordyceps by using morels.
  • Cordyceps sinensis is a complex of larvae of the ergot fungus Cordyceps sinensis parasitic on the larvae of Helicoptera insects and the corpse of the larvae. It is a precious nourishing Chinese medicinal material with functions of regulating the immune system, anti-tumor, anti-fatigue, anti-aging and other functions. .
  • the consumption of Cordyceps has been increasing year by year, but the problem of excessive heavy metals in wild Cordyceps is common. For example, the As, Hg, Pb, Cd, Cu and other heavy metal contents of 45 Cordyceps samples from different commercial sources were measured.
  • the current method to reduce the heavy metals in the breeding of Cordyceps is to acidify the epidermis of the Cordyceps, and then rinse the heavy metals decomposed by acidification with pure water, but this method may damage the integrity and quality of the Cordyceps.
  • the treatment of soil heavy metal pollution will be more conducive to protecting the quality of Cordyceps sinensis.
  • soil restoration methods mainly include physical restoration, chemical restoration, agricultural restoration, and bioremediation.
  • Bioremediation also includes phytoremediation, animal restoration, and microbial restoration.
  • Mushrooms are used at home and abroad. There are many studies on the restoration of soil heavy metal pollution, and it is found that mushrooms have strong tolerance and enrichment ability to heavy metals. The mushrooms can directly absorb heavy metals floating in the air or accumulate heavy metals in the soil matrix through mycelium.
  • Morchella (Morchella deliciosa Fr.), low-temperature fungus, belongs to the fungus kingdom, Ascomycota, Ascomycetes, Pelicomycetes, Morchella family, Morchella genus, also known as Grass Lizhu, belonging to cold Cool edible (medicinal) fungus is a precious edible and medicinal fungus.
  • the purpose of the present invention is to provide a method for repairing soil pollution to reduce the excess of arsenic and heavy metals in the ecologically breeding Cordyceps sinensis.
  • the purpose of the present invention is achieved through the following technical solutions:
  • Morel as the main enrichment material for soil heavy metals, especially arsenic ions, and make full use of the similar growth environment of Morel and Cordyceps sinensis. They like low temperature and can grow in the soil. The growth cycle is short. The cultivation technology is mature. The ability to accumulate heavy metals has the characteristics of preference for arsenic enrichment. Morels are cultivated in the habitat where Cordyceps sinensis is bred, and morels are harvested through batch cultivation to continuously dilute the heavy metals in the soil and water, so as to compete with Cordyceps sinensis.
  • the host and the host feed competed until the Cordyceps was unearthed, and the heavy metal content of Cordyceps sinensis without morel treatment and after morel treatment, and the heavy metal content of the background soil before and after the treatment were determined.
  • the use of a grid division device facilitates the uniform removal of arsenic and heavy metals and the increase in the output of Cordyceps sinensis.
  • the invention specifically provides a method for interplanting morels and cordyceps. .
  • a layer of 3-5cm sterile first humus soil is spread on the bottom layer, the first feed is placed on the first humus soil layer, and the morel mushrooms are evenly placed on the first feed , Cover the first humus soil 2-8cm, plant the Cordyceps sinensis host eggs on the surface of the humus soil, and control the cultivation under the first culture condition.
  • a layer of sterilized first humus soil of 3-5cm is spread on the bottom layer of the dividing device with a cell side length of 15cm*15cm, the first feed is placed on the first humus soil layer, and the Morel fungus clumps are evenly placed on the first feed, covering 2-8 cm of the first humus soil, and the host eggs of Cordyceps sinensis are planted on the surface of the humus soil to control the cultivation under the first culture condition.
  • the first humus soil is the humus soil on which more than 2 stubbles of morels are continuously cultivated.
  • the sterilization method of the humus is high-pressure steam and heat sterilization, specifically, using 0.15MPa, 121°C high-pressure steam sterilization for 30 minutes, and then oven at 50-90°C overnight.
  • the host eggs of the Cordyceps sinensis are the eggs of Hepidia.
  • the first feed is tuber roots such as Potentilla, small rhubarb, Polygonum vulgare, Salvia miltiorrhiza, grass silkworm, carrot or Jerusalem artichoke.
  • the infection material in the infection treatment includes 40-100 ascospores/ml, 20-70 conidia/ml, and mycelium 0.1-1 mg/ml.
  • the first culture condition and the second culture condition are that the soil humidity is 40-70%, the culture temperature is 6-18°C, and the culture needs to be given light Stimulating, 4-12 hours of light per day.
  • a method comprising the following steps can be used to cultivate Morchella: strain preparation, indoor cultivation and subsequent processing steps.
  • strains Preparation of strains: mix sawdust, bran, phosphate fertilizer, gypsum, humus soil and water, mix well, pile up and ferment, fill with polyethylene plastic bags, sterilize, and then enter morel strains. Adopt the two-head inoculation method, seal the bag mouth, cultivate until the hyphae is full, and then take off the bag to obtain morel sticks for cultivation.
  • Subsequent treatment The morels are harvested at maturity, and the humus soil is collected and sterilized to continue the cultivation of the next crop of morels.
  • the first humus soil is obtained through the following steps:
  • the first humus soil is obtained through the following steps:
  • the first humus soil is obtained through the following steps:
  • the content of arsenic and heavy metals in the soil was determined by atomic fluorescence method GB/T 22105-2008.
  • the content of arsenic and heavy metals in Morchella and Cordyceps was determined by inductively coupled plasma mass spectrometry.
  • Heavy metals refer to mercury, lead, copper, etc.
  • the present invention uses Morchella as a heavy metal bioaccumulator in the breeding of Cordyceps sinensis. It makes full use of the similar growth characteristics of the two, similar temperature, humidity and illumination requirements, and their respective ability to enrich heavy metals, especially the effect of Morchella on arsenic and heavy metals.
  • the characteristics of good enrichment ability of Cordyceps sinensis, interplanting morel mushrooms while breeding Cordyceps sinensis, are harvested in batches to continuously dilute the heavy metals in the soil and water, thereby reducing the heavy metal content of Cordyceps sinensis.
  • the content of arsenic and heavy metals in Cordyceps bred after morel treatment is significantly lower than that of Cordyceps bred separately.
  • the application of this interplanting method in the cell division device has more obvious effects.
  • the arsenic and heavy metals in the cultivated Cordyceps are The content is significantly reduced, and the output is also increased.
  • Figure 1 is a grid diagram of the grid division device, in which the length, width and height of each small grid is 15cm*15cm*30cm;
  • Figure 2 is an external block diagram of the grid division device
  • Fig. 3 is a perspective view of the grid dividing device.
  • the humus soil from the source of Cordyceps was collected, passed through a 10-mesh sieve, and uniformly adjusted to a humidity of 40-70% after autoclave sterilization and heat sterilization.
  • Atomic fluorescence method GB/T 22105-2008 was used to determine arsenic, mercury, and lead in the background soil.
  • the content of equal heavy metals, the measured content of As is 13.60mg/kg, Hg is 0.036mg/kg, and Pb is 16.69mg/kg.
  • Example 1 The absorption of heavy metal ions by Morchella in the soil with excessive arsenic
  • the temperature in the clean indoor cultivation area is 6-18°C, and the light is 4-12 hours a day.
  • Each bed surface is covered with 4cm of over-standard soil. After spreading, the sticks that have been removed from the plastic bag are broken into 5cm-sized bacterial clumps one by one. On the bed surface, 2 bacteria bags can be used per square meter of the bed surface. Cover soil with 5cm to maintain soil moisture content of 40-70%.
  • Morel mushrooms are harvested after maturity, calculated based on 180kg of fresh mushrooms per mu, the fruit body is 215mg/kg as determined by inductively coupled plasma mass spectrometry "Chinese Pharmacopoeia” 2010 edition appendix, and the average annual harvest is 5 -6 times, the average concentration of arsenic after one year is 193.5g, and the soil arsenic removal rate can reach more than 50%.
  • Example 2 Using morel and Cordyceps interplanting to compete with heavy metal ions in background soil
  • Each bed surface is covered with 3cm first humus soil, on which are the roots of Potentilla, small rhubarb, Polygonum vulgare, Salvia miltiorrhiza, grass silkworm, carrot or Jerusalem artichoke, etc.
  • the 6cm size morel mushroom block Place it evenly on the feed tuber, cover 4cm of soil, and plant the Hepialidae eggs on the topsoil layer to maintain the soil moisture content of 40-70%.
  • Example 3 Interplanting Morel and Cordyceps to compete with heavy metal ions in background soil
  • Each bed surface is covered with 5cm of the first humus soil, on which are the roots of Potentilla, small rhubarb, Polygonum vulgare, Salvia, grass silkworm, carrot, Jerusalem artichoke, etc. At the same time, 3cm size morel mushroom block Place them evenly on the feed tuber roots, cover 6cm of soil, and plant the Hepidia henryi eggs on the topsoil layer to maintain the soil moisture content of 40-70%.
  • Example 4 Interplanting Morel and Cordyceps to compete with heavy metal ions in background soil
  • Each bed surface is covered with 4cm of first humus soil, on which are 4cm of roots such as fern, small rhubarb, Polygonum vulgare, salvia, grass silkworm, carrot, Jerusalem artichoke, etc., and 5cm of morel
  • the clumps are evenly placed on the roots of the feed, covered with 5cm of soil, and the hepidia eggs are sown on the topsoil layer to maintain the soil moisture content of 40-70%.
  • Example 5 Interplanting Morel and Cordyceps to compete with heavy metal ions in background soil
  • Each bed surface is covered with 4cm of humus soil, and 4cm of roots such as fern, small rhubarb, Polygonum vulgare, salvia, grass silkworm, carrot, Jerusalem artichoke, etc. are covered on it, and the 5cm size of Morel fungus is evenly distributed. Put it on the feed, cover the soil with 5cm, and plant the bat moth eggs on the topsoil layer to maintain the soil moisture content of 40-70%, the temperature of the clean area is 6-18 °C, and the light is 8 hours a day.
  • Step 3) Harvest the Morchella fruiting bodies again, pick out the bat moths and feed roots, and uniformly autoclave and heat sterilize the remaining parts to form the third humus soil with a humidity of 40-70%. Step 3) was repeated again, after 6 consecutive cultivations of Morchella, after the Cordyceps was unearthed, the content of heavy metals in Cordyceps was determined. The content of arsenic and heavy metals, and the yield of Cordyceps were equivalent to those in Example 4.
  • Example 6 The absorption of heavy metal ions by morel planting in a grid device in the soil with excessive arsenic
  • the indoor cultivation clean area has a temperature of 6-18°C, light for 4-12 hours a day, and the planting bed surface is a grid device with a length and width of 15cm*15cm per cell (as shown in Figure 1-3), that is, the grid bed surface .
  • Each bed surface is covered with 4cm of extra-standard soil. After spreading, the bacteria sticks that have been removed from the plastic bag are broken into 5cm-sized bacteria blocks and placed on the bed surface. 2 bacteria bags per square meter of bed surface can be used. Cover soil with 5cm to maintain soil moisture content of 40-70%.
  • Morel mushrooms are harvested after maturity, and the fruit bodies are collected by inductively coupled plasma mass spectrometry "Chinese Pharmacopoeia” 2010 edition appendix to determine the content of arsenic. The average annual harvest is 5-6 times, and the average concentration of arsenic after one year is determined. The amount is compared with the morel grown on the bed surface without the compartment device in Example 1, as shown in Table 4.
  • the temperature of the clean area is 6-18°C
  • the light is 8 hours a day
  • the planting bed surface is a grid device with a length and width of 15cm*15cm per small grid (as shown in Figure 1-3), that is, the grid bed surface.
  • Each bed surface is covered with 4cm of extra-standard soil. After spreading, the bacteria sticks that have been removed from the plastic bag are broken into 5cm-sized bacteria blocks and placed on the bed surface. 2 bacteria bags per square meter of bed surface can be used. Cover soil with 5cm to maintain soil moisture content of 40-70%.
  • Each layer of the grid bed is covered with 4cm of the first humus soil, on which are 4cm of fern, small rhubarb, Polygonum vulgare, salvia, grass silkworm, carrot, Jerusalem artichoke and other roots.
  • 4cm sheep The fungus clumps are evenly placed on the roots of the feed, covered with 5cm soil, and the hepidia corpus eggs are sown on the topsoil layer to maintain the soil moisture content of 40-70%.

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Abstract

一种利用羊肚菌降低生态繁育冬虫夏草砷及重金属超标的方法，包括将冬虫夏草和羊肚菌套种，利用羊肚菌和冬虫夏草的生长特性相似，羊肚菌对砷及重金属的吸收能力强等特点，从土壤中富集重金属，从而降低冬虫夏草全草中的砷及重金属含量。

Description

一种降低生态繁育冬虫夏草砷及重金属含量的方法 技术领域
本发明涉及通过微生物修复降低土壤污染繁育优质冬虫夏草技术领域，具体涉及一种利用羊肚菌降低生态繁育冬虫夏草砷及重金属超标的方法。
背景技术
冬虫夏草是麦角菌科真菌冬虫夏草菌寄生在蝙蝠蛾科昆虫幼虫上的子座及幼虫尸体的复合体，是一种名贵滋补中药材，具有调节免疫系统功能，抗肿瘤、抗疲劳、抗衰老等功效。近年随着人们生活水平的提高，保健观念不断增强，冬虫夏草的消费量逐年增加，但野生冬虫夏草普遍存在重金属超标问题。例如对45个不同市售来源的冬虫夏草样品测定其As、Hg、Pb、Cd、Cu等重金属含量，在45个样品中，As含量超标率达到100％，Hg含量超标率也达到70％，其他3种重金属均未检出超标现象。长期食用冬虫夏草、冬虫夏草粉及纯粉片等产品会造成砷汞的过量摄入，并可能在人体内蓄积，存在较高风险。
野生冬虫夏草重金属超标是同其复杂的生活史密切相关的，冬虫夏草生活史须经历无性阶段和有性阶段，此过程绝大多数时间都是在土壤中完成的，因此土壤是造成冬虫夏草重金属超标的主要来源之一。现有的生态繁育冬虫夏草项目同野生虫草一样，同样采用高原土壤作为冬虫夏草寄主昆虫的生境，故生态繁育冬虫夏草仍存在重金属超标的风险。目前降低繁育冬虫夏草重金属的方法是对冬虫夏草表皮进行酸化，再用纯水冲洗掉酸化分解出来的重金属，但这种方法对冬虫夏草处理，可能会破坏冬虫夏草的完整性和品质。从冬虫夏草的生长环境出发，治理土壤重金属污染，会更利于保护冬虫夏草的品质。
土壤重金属污染治理和恢复的难度较大，目前土壤修复的方式主要有物理修复、化学修复、农业修复和生物修复等，生物修复又包括植物修复和动物修复以及微生物修复，其中国内外利用蕈菌修复土壤重金属的污染研究较多，发现蕈菌对重金属有较强的耐受和富集能力，蕈菌可以通过直接吸收空气中漂浮的重金属或通过菌丝体累积土壤基质中的重金属。
羊肚菌(Morchella deliciosa Fr.)，喜低温蕈菌，隶属真菌界，子囊菌门，子囊菌纲，盘菌目，羊肚菌科，羊肚菌属，又名草笠竹，属于喜冷凉型食(药)用菌，是一种珍贵的食用菌和药用菌。
发明内容
本发明的目的在于提供一种修复土壤污染来降低生态繁育冬虫夏草中砷及重金属超标的 方法。本发明的目的通过以下技术方案来实现：
采用羊肚菌作为土壤重金属尤其是砷离子的主要富集材料，充分利用羊肚菌与冬虫夏草生长环境相似都喜低温，且能在土壤中生长，生长周期较短，栽培技术成熟，羊肚菌富集重金属能力尤其对砷富集具有偏好性等特点，在繁育冬虫夏草的生境中同时栽培羊肚菌，并通过分批培养采收羊肚菌，不断稀释土壤及水分当中的重金属，从而与冬虫夏草寄主及寄主饲料形成竞争，直到虫草出土，测定未经过羊肚菌处理和经过羊肚菌处理的冬虫夏草重金属含量，以及处理前后背景土壤的重金属含量。同时，采用分格装置利于砷及重金属的均匀去除以及冬虫夏草产量的提升。
本发明具体提供一种羊肚菌和冬虫夏草套种方法。。
优选的，在底层铺一层3-5cm的灭菌的第一腐殖土，将第一饲料置于所述第一腐殖土层上，将羊肚菌菌块均匀置于第一饲料上，覆盖第一腐殖土2-8cm，在腐殖土表层播植冬虫夏草寄主卵，控制第一培养条件培养。
优选的，在单元格边长为15cm*15cm的分格装置底层铺一层3-5cm的灭菌的第一腐殖土，将第一饲料置于所述第一腐殖土层上，将羊肚菌菌块均匀置于第一饲料上，覆盖第一腐殖土2-8cm，在腐殖土表层播植冬虫夏草寄主卵，控制第一培养条件培养。
羊肚菌成熟后收割羊肚菌，分离出孵化的冬虫夏草寄主虫以及第一饲料，收集剩余部分混合均匀灭菌后得到第二腐殖土，分离出的第一饲料形成第二饲料，分离出的冬虫夏草寄主虫侵染处理。在底层铺一层3-5cm的第二腐殖土，其上铺放第二饲料，再覆盖第二腐殖土2-8cm，接入已侵染处理的冬虫夏草寄主虫，控制第二培养条件继续培养直至冬虫夏草长出子座并成熟。
上述方法中，第一腐殖土为连续栽培2茬以上羊肚菌的腐殖土。
上述方法中，腐殖土的灭菌方法为高压蒸汽和加热灭菌，具体是采用0.15MPa，121℃的高压蒸汽灭菌30min，然后烘箱50-90℃，过夜。
上述方法中，冬虫夏草寄主卵为蝙蝠蛾虫卵。
上述方法中，第一饲料为蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜或菊芋等块根。
上述方法中，侵染处理中的侵染材料包含子囊孢子40-100个/ml，分生孢子20-70个/ml，菌丝体0.1-1mg/ml。
上述方法中，由于冬虫夏草和羊肚菌的生长特性相似，所述第一培养条件和第二培养条件为土壤湿度为40-70％，培养温度为6-18℃，同时的，培养需给予光照刺激，光照时间4-12小时每天。
上述方法中，为了羊肚菌更好的生长充分吸收土壤中重金属，将羊肚菌菌丝体掰成3-7cm 大小菌块。
上述方法中，为了获得第一腐殖土，可采用包含如下步骤的方法栽培羊肚菌：菌种制备、室内栽培和后续处理步骤。
菌种的制备：木屑、麸皮、磷肥、石膏、腐殖土和水混合拌好，拌好后堆积发酵，用聚乙烯塑料袋装料，灭菌，即可接入羊肚菌菌种，采用两头接种法，封好袋口，培养至菌丝满袋后脱袋得羊肚菌菌棒即可栽培。
室内栽培：每层床面上覆盖3-5cm的腐殖土，铺平后将上述菌棒逐个掰成3-7cm大小菌块铺在腐殖土上，再覆盖腐殖土2-8cm，控制培养条件进行培养。
后续处理：羊肚菌成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的栽培。
重复上述步骤2次及以上，获得第一腐殖土。
任选的，所述第一腐殖土通过如下步骤获得：
1)将50％-85％木屑、15％-40％麸皮、0.3％-2％磷肥、0.3％-2％石膏、1％-5％腐殖土和水混合拌好，料水比为1:1.1-1:1.6，堆积发酵后，装料灭菌，接入羊肚菌菌种，封好袋口，培养至菌丝满袋，一段时间后脱袋得羊肚菌菌棒；
2)在腐殖土上铺放脱袋的羊肚菌菌棒，覆盖腐殖土，维持土壤含水量，培养温度6-18℃，光照每天4-12小时，培养；
3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的栽培；
4)重复上述步骤2次及以上，获得第一腐殖土。
任选的，所述第一腐殖土通过如下步骤获得：
1)将60％-85％木屑、10％-35％麸皮、0.5％-1.5％磷肥、0.5％-1.5％石膏、1％-5％腐殖土和水混合拌好，料水比为1:1.2-1:1.5，堆积发酵后，装料灭菌，接入羊肚菌菌种，封好袋口，培养至菌丝满袋，一段时间后脱袋；
2)在腐殖土上铺放脱袋的羊肚菌菌棒，覆盖腐殖土，维持土壤含水量，控制培养温度6-18℃，光照每天4-12小时，培养；
3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的栽培；
4)重复上述步骤4次及以上，获得第一腐殖土。
任选的，所述第一腐殖土通过如下步骤获得：
1)将75％木屑、20％麸皮、1％磷肥、1％石膏、3％腐殖土和水混合拌好，料水比为1:1.3，堆积发酵20天后，装料100℃条件下灭菌8小时，接入羊肚菌菌种，封好袋口，培养至菌丝满袋，一段时间后脱袋；
2)将羊肚菌菌棒分割成3-7cm的菌块，在腐殖土上铺放所述菌块，覆盖腐殖土，维持土 壤含水量，控制培养温度6-18℃，光照每天4-12小时，培养；
3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的栽培；
4)重复上述步骤5次，获得第一腐殖土。上述羊肚菌和冬虫夏草套种方法所取得的有益效果是，冬虫夏草中砷及重金属含量要比直接采用虫草原地的腐殖土繁育的冬虫夏草或野生冬虫夏草中的砷及重金属含量都明显降低。
土壤中砷及重金属含量采用原子荧光法GB/T 22105-2008测定。
羊肚菌和冬虫夏草砷及重金属含量采用电感耦合等离子体质谱法测定。
重金属是指汞、铅、铜等。
本发明的优点：
本发明在繁育冬虫夏草时以羊肚菌作为重金属的生物富集器，充分利用了两者生长特性相似，温度湿度光照要求相似，各自对重金属富集能力不同，尤其是羊肚菌对砷及重金属的富集能力好的特点，在繁育冬虫夏草的同时套种羊肚菌，通过分批采收，不断稀释土壤及水分当中的重金属，从而降低冬虫夏草的重金属含量。经过羊肚菌处理繁育出的冬虫夏草，相比单独繁育的冬虫夏草，其砷及重金属含量明显降低，将该套种方法应用在分格装置中，效果更明显，所培育出的冬虫夏草中的砷及重金属含量明显降低，产量也有所提升。
附图说明
图1是分格装置内格图，其中每个小格子长宽高为15cm*15cm*30cm；
图2是分格装置外框图；
图3是分格装置立体图。
具体实施方式
为能进一步解释本发明的特征、技术手段以及所达到的具体目的、功能，解析本发明的优点与方案，结合以下具体实施方式对本发明的详述进行进一步地解释。应理解，以下实施例仅用于说明本发明而不用于限制本发明的保护范围。此外应理解，在阅读了本发明的内容后，本领域技术人员可以对本发明做各种修改和改动，这些等价形式同样落于本发明所要求保护的范围内。
采集虫草源地腐殖土，过10目筛，高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％，采用原子荧光法GB/T 22105-2008测定背景土壤中砷及汞、铅等重金属含量，测得各自含量As为13.60mg/kg，Hg为0.036mg/kg，Pb为16.69mg/kg。
实施例1：羊肚菌在砷超标土壤中对重金属离子的吸收
1)砷超标土壤的配制采用亚砷酸钠将背景土壤中砷含量设置为120mg/kg(国标<40mg/kg)
2)菌种制备配方：木屑75％、麸皮20％、磷肥1％、石膏1％、腐殖土3％；料水比1:1.3，拌好后堆积发酵20天，含水量60％。采用17×33cm聚乙烯塑料袋装料，每袋500-600克，在100℃条件下灭菌8小时，即可接入菌种。采用两头接种法，封好袋口，置于22-25℃温度下培养30天。菌丝满袋后5-6天，即可栽培。
3)室内栽培洁净区温度6-18℃，光照每天4-12小时，每层床面上覆盖4cm的超标土壤，铺平后将脱去塑料袋的菌棒逐个掰成5cm大小菌块铺在床面上，每平方米床面可用菌袋2个。覆土5cm，维持土壤含水量40-70％。
4)羊肚菌菇成熟后采收，按单次亩产鲜菇180kg计算，子实体采用电感耦合等离子体质谱法《中国药典》2010版附录测定砷含量为215mg/kg，年均采收5-6次，一年后对砷的平均富集量是193.5g，土壤砷去除率可达50％以上。
实施例2：利用羊肚菌和冬虫夏草套种竞争背景土壤中重金属离子
1)将木屑61％、麸皮30％、磷肥2％、石膏2％、腐殖土5％混合，加水，料水比1:1.2，拌好后堆积发酵20天，含水量60％。采用17×33cm聚乙烯塑料袋装料，每袋600克，在100℃条件下灭菌8小时，即可接入羊肚菌菌种。采用两头接种法，封好袋口，置于23℃温度下培养30天。菌丝满袋后5-6天，即可栽培。
2)每层床面上覆盖3cm的腐殖土，铺平后将脱去塑料袋的羊肚菌菌棒逐个掰成6cm大小菌块铺在床面上，覆土4cm，维持土壤含水量40-70％，洁净区温度6-18℃，光照每天6小时。
3)羊肚菌成熟后开始采收，收集腐殖土继续用于下一茬羊肚菌的培养，重复上述步骤2)，直至得到栽培了4茬羊肚菌的腐殖土，高温高压和加热灭菌后即为第一腐殖土。
4)每层床面上覆盖3cm的第一腐殖土，其上是蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜或菊芋等块根，同时将6cm大小的羊肚菌菌块均匀置于饲料块根之上，覆土4cm，蝙蝠蛾虫卵播植于表土层之上，维持土壤含水量40-70％。
5)将成熟后的套种羊肚菌收割，挑出蝙蝠蛾虫以及蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜或菊芋等块根，收集剩余部分混合均匀高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％的第二腐殖土，将蝙蝠蛾虫侵染处理；每层床面上覆盖3cm的第二腐殖土，其上重 铺上述饲料块根，覆盖第二腐殖土4cm，接入侵染处理的蝙蝠蛾幼虫。
6)控制洁净区温度6-18℃，光照每天6小时，培养直至冬虫夏草出土。
实验中设定了单独繁育的冬虫夏草的对照组(相同培养条件下用背景土壤繁育的冬虫夏草)，野生组(虫草源地野生的冬虫夏草)，分别测定了对照组、野生组、本实施例处理组中冬虫夏草的重金属含量以及产量，如表1。处理组的冬虫夏草砷及重金属含量明显低于对照草和野生草。
表1 虫草重金属含量及产量
Figure PCTCN2020079156-appb-000001
实施例3：利用羊肚菌和冬虫夏草套种竞争背景土壤中重金属离子
1)将木屑65％、麸皮30％、磷肥0.5％、石膏0.5％、腐殖土4％混合，加水，料水比1:1.5，拌好后堆积发酵20天，含水量60％。采用17×33cm聚乙烯塑料袋装料，每袋600克，在100℃条件下灭菌8小时，即可接入羊肚菌菌种。采用两头接种法，封好袋口，置于25℃温度下培养30天。菌丝满袋后5-6天，即可栽培。
2)每层床面上覆盖5cm的腐殖土，铺平后将脱去塑料袋的羊肚菌菌棒逐个掰成3cm大小菌块铺在床面上，覆土6cm，维持土壤含水量40-70％，洁净区温度6-18℃，光照每天10小时。
3)羊肚菌成熟后开始采收，收集腐殖土继续用于下一茬羊肚菌的培养，重复上述步骤2)，直至得到栽培了5茬羊肚菌的腐殖土，高温高压和加热灭菌后即为第一腐殖土。
4)每层床面上覆盖5cm的第一腐殖土，其上是蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜、菊芋等块根，同时将3cm大小的羊肚菌菌块均匀置于饲料块根之上，覆土6cm，蝙蝠蛾虫卵播植于表土层之上，维持土壤含水量40-70％。
5)收割成熟的套种羊肚菌，挑出蝙蝠蛾虫以及饲料块根，收集剩余部分混合均匀高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％的第二腐殖土，每层床面上覆盖5cm的第二腐殖土，其上重铺上述饲料块根，覆盖第二腐殖土6cm，接入侵染处理的蝙蝠蛾幼虫。
6)控制洁净区温度6-18℃，光照每天10小时，培养直至冬虫夏草出土。
实验中设定了单独繁育的冬虫夏草的对照组(用背景土壤繁育的冬虫夏草)，野生组(虫草源地野生的冬虫夏草)，分别测定了对照组、野生组、本实施例处理组中冬虫夏草的重金属 含量以及产量，如表2。处理组的冬虫夏草砷及重金属含量明显低于对照草和野生草。
表2 虫草重金属含量及产量
Figure PCTCN2020079156-appb-000002
实施例4：利用羊肚菌和冬虫夏草套种竞争背景土壤中重金属离子
1)将木屑75％、麸皮20％、磷肥1％、石膏1％、腐殖土3％混合，加水，料水比1:1.3，拌好后堆积发酵20天，含水量60％。采用17×33cm聚乙烯塑料袋装料，每袋500克，在100℃条件下灭菌8小时，即可接入羊肚菌菌种。采用两头接种法，封好袋口，置于25℃温度下培养30天。菌丝满袋后5-6天，即可栽培。
2)每层床面上覆盖4cm的腐殖土，铺平后将脱去塑料袋的羊肚菌菌棒逐个掰成5cm大小菌块铺在床面上，覆土5cm，维持土壤含水量40-70％，洁净区温度6-18℃，光照每天8小时。
3)羊肚菌成熟后开始采收，收集腐殖土继续用于下一茬羊肚菌的培养，重复上述步骤2)，直至得到栽培了5茬羊肚菌的腐殖土，高温高压和加热灭菌后即为第一腐殖土。
4)每层床面上覆盖4cm的第一腐殖土，其上是4cm的蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜、菊芋等块根，同时将5cm大小的羊肚菌菌块均匀置于饲料块根之上，覆土5cm，蝙蝠蛾虫卵播植于表土层之上，维持土壤含水量40-70％。
5)收割成熟的套种羊肚菌，挑出蝙蝠蛾虫以及饲料块根，收集剩余部分混合均匀高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％的第二腐殖土，每层床面上覆盖4cm的第二腐殖土，其上重铺上述饲料块根，覆盖第二腐殖土5cm，接入侵染处理的蝙蝠蛾幼虫。
6)控制洁净区温度6-18℃，光照每天8小时，培养直至冬虫夏草出土。
实验中设定了单独繁育的冬虫夏草的对照组(用背景土壤繁育的冬虫夏草)，野生组(虫草源地野生的冬虫夏草)，分别测定了对照组、野生组、本实施例处理组中冬虫夏草的重金属含量以及产量，如表3。处理组的冬虫夏草砷及重金属含量明显低于对照草和野生草。
表3 虫草重金属含量及产量
Figure PCTCN2020079156-appb-000003
实施例5：利用羊肚菌和冬虫夏草套种竞争背景土壤中重金属离子
1)每层床面上覆盖4cm的腐殖土，其上是4cm的蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜、菊芋等块根，将5cm大小的羊肚菌菌块均匀置于饲料之上，覆土5cm，蝙蝠蛾虫卵播植于表土层之上，维持土壤含水量40-70％，洁净区温度6-18℃，光照每天8小时。
2)将成熟的套种羊肚菌收割，挑出蝙蝠蛾虫以及饲料块根，收集剩余部分混合均匀高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％的第二腐殖土，将蝙蝠蛾幼虫侵染处理。
3)每层床面上覆盖4cm的第二腐殖土，将步骤2)挑出的饲料块根置于第二腐殖土之上，同时将新一批5cm大小的羊肚菌菌块均匀置于所述饲料块根之上，覆土5cm，接入侵染处理过的蝙蝠蛾幼虫。控制洁净区温度6-18℃，光照每天8小时。
4)再采收一次羊肚菌子实体，并将蝙蝠蛾虫和饲料块根挑出，将剩余部分均匀高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％的第三腐殖土，再次重复步骤3)，连续栽培6次羊肚菌后，待虫草出土，测定虫草重金属含量，其砷和重金属含量，以及冬虫夏草产量与实施例4相当。
实施例6：分格装置种植羊肚菌在砷超标土壤中对重金属离子的吸收
1)采用亚砷酸钠将背景土壤中砷含量设置为120mg/kg。
2)菌种制备配方：木屑75％、麸皮20％、磷肥1％、石膏1％、腐殖土3％；料水比1:1.3，拌好后堆积发酵20天，含水量60％。采用17×33cm聚乙烯塑料袋装料，每袋500-600克，在100℃条件下灭菌8小时，即可接入菌种。采用两头接种法，封好袋口，置于22-25℃温度下培养30天。菌丝满袋后5-6天，即可栽培。
3)室内栽培洁净区温度6-18℃，光照每天4-12小时，种植床面为每小格长宽15cm*15cm的分格装置(如图1-3所示)，即分格床面。每层床面上覆盖4cm的超标土壤，铺平后将脱去塑料袋的菌棒逐个掰成5cm大小菌块铺在床面上，每平方米床面可用菌袋2个。覆土5cm，维持土壤含水量40-70％。
4)羊肚菌菇成熟后采收，子实体采用电感耦合等离子体质谱法《中国药典》2010版附录测定砷含量年均采收5-6次，并测定一年后对砷的平均富集量，与实施例1中不用分格装置也即不分格床面培植的羊肚菌进行比较，见表4。
表4 羊肚菌子实体产量及As含量
Figure PCTCN2020079156-appb-000004
实施例7：分格装置套种羊肚菌和冬虫夏草竞争背景土壤中重金属离子
1)将木屑75％、麸皮20％、磷肥1％、石膏1％、腐殖土3％；料水比1:1.3，拌好后堆积发酵20天，含水量60％。采用17×33cm聚乙烯塑料袋装料，每袋500克，在100℃条件下灭菌8小时，即可接入羊肚菌菌种。采用两头接种法，封好袋口，置于25℃温度下培养30天。菌丝满袋后5-6天，即可栽培。
2)洁净区温度6-18℃，光照每天8小时，种植床面为每小格长宽15cm*15cm的分格装置(如图1-3所示)，即分格床面。每层床面上覆盖4cm的超标土壤，铺平后将脱去塑料袋的菌棒逐个掰成5cm大小菌块铺在床面上，每平方米床面可用菌袋2个。覆土5cm，维持土壤含水量40-70％。
3)羊肚菌成熟后开始采收，收集腐殖土继续用于下一茬羊肚菌的培养，重复上述步骤2)，直至得到栽培了5茬羊肚菌的腐殖土，高温高压和加热灭菌后即为第一腐殖土。
4)每层分格床面上覆盖4cm的第一腐殖土，其上是4cm的蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜、菊芋等块根，同时将5cm大小的羊肚菌菌块均匀置于饲料块根之上，覆土5cm，蝙蝠蛾虫卵播植于表土层之上，维持土壤含水量40-70％。
5)收割成熟的套种羊肚菌，挑出蝙蝠蛾虫以及饲料块根，收集剩余部分混合均匀高压蒸汽灭菌和加热灭菌后统一调配成湿度40-70％的第二腐殖土，每层床面上覆盖4cm的第二腐殖土，其上重铺上述饲料块根，覆盖第二腐殖土5cm，接入侵染处理的蝙蝠蛾幼虫。
6)控制洁净区温度6-18℃，光照每天8小时，培养直至冬虫夏草出土。
实验中设定了分格床面条件下繁育的冬虫夏草对照组(相同培养条件下用背景土壤繁育的冬虫夏草)，野生组(虫草源地野生的冬虫夏草)，分别测定了分格对照组、野生组、分格处理组(本实施例)中冬虫夏草的重金属含量以及产量，与不分格对照组(即实施例4)进行比较，如表5。本实施例分格处理组的冬虫夏草砷及重金属含量明显低于对照草和野生草。
表5 虫草重金属含量及产量
Figure PCTCN2020079156-appb-000005
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种降低生态繁育冬虫夏草砷及重金属含量的方法，其特征在于将羊肚菌和冬虫夏草套种。
  2. 根据权利要求1所述的方法，其特征在于包括如下步骤：
    1)在第一腐殖土层上铺放第一饲料；
    2)将羊肚菌菌块均匀置于所述饲料之上，再覆盖所述第一腐殖土，在腐殖土表层播植蝙蝠蛾虫卵，控制第一培养条件培养；
    3)待所述羊肚菌菌块上长出的羊肚菌成熟后，收割所述羊肚菌，分离出已孵化的蝙蝠蛾幼虫以及所述第一饲料，将剩余部分混合均匀灭菌形成第二腐殖土，分离出的第一饲料形成第二饲料，分离出的蝙蝠蛾幼虫侵染处理；
    4)在所述第二腐殖土层上铺放第二饲料，再覆盖所述第二腐殖土，将已侵染处理过的蝙蝠蛾幼虫置于其上，控制第二培养条件继续培养得冬虫夏草；
    优选的，在步骤1)中，将所述第一腐殖土层铺在分格装置的底层。
  3. 根据权利要求2所述的方法，所述第一腐殖土为对连续栽培2茬以上羊肚菌的腐殖土灭菌所得；所述第一腐殖土层和所述第二腐殖土层的厚度为3-5cm，所述第一腐殖土和所述第二腐殖土的覆土厚度为2-8cm。
  4. 根据权利要求2所述的方法，所述第一饲料为蕨麻、小大黄、珠芽蓼、丹参、草石蚕、胡萝卜或菊芋等植物块根。
  5. 根据权利要求2所述的方法，所述侵染处理中的侵染材料包含子囊孢子40-100个/ml，分生孢子20-70个/ml，菌丝体0.1-1mg/ml。
  6. 根据权利要求2所述的方法，所述第一培养条件和所述第二培养条件为土壤含水量40-70％，温度6-18℃，光照时间4-12小时每天。
  7. 根据权利要求2所述的方法，所述羊肚菌菌块直径为3-7cm。
  8. 根据权利要求2所述的方法，所述分格装置的每小格长宽为15cm*15cm。
  9. 根据权利要求2-8任一项所述的方法，所述第一腐殖土通过如下步骤获得：
    1)将木屑、麸皮、磷肥、石膏、腐殖土和水混合拌好，堆积发酵后，装料灭菌，接入羊肚菌菌种，封好袋口，培养至菌丝满袋后脱袋得羊肚菌菌棒；
    2)在腐殖土上铺放所述羊肚菌菌棒，再覆盖腐殖土，维持土壤含水量进行培养；
    3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的培养；
    4)重复上述步骤2次及以上，获得第一腐殖土；
    任选的，所述第一腐殖土通过如下步骤获得：
    1)将50％-85％木屑、15％-40％麸皮、0.3％-2％磷肥、0.3％-2％石膏、1％-5％腐殖 土和水混合拌好，料水比为1:1.1-1:1.6，堆积发酵后，装料灭菌，接入羊肚菌菌种，封好袋口，培养至菌丝满袋，一段时间后脱袋；
    2)在腐殖土上铺放脱袋的羊肚菌菌棒，覆盖腐殖土，控制培养条件，培养；
    3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的培养；
    4)重复上述步骤2次及以上，获得第一腐殖土；
    任选的，所述第一腐殖土通过如下步骤获得：
    1)将60％-85％木屑、10％-35％麸皮、0.5％-1.5％磷肥、0.5％-1.5％石膏、1％-5％腐殖土和水混合拌好，料水比为1:1.2-1:1.5，堆积发酵后，装料灭菌，接入羊肚菌菌种，封好袋口，培养至菌丝满袋，一段时间后脱袋；
    2)在腐殖土上铺放脱袋的羊肚菌菌棒，覆盖腐殖土，维持土壤含水量40-70％，控制培养温度6-18℃，光照每天4-12小时，培养；
    3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的培养；
    4)重复上述步骤4次及以上，获得第一腐殖土；
    任选的，所述第一腐殖土通过如下步骤获得：
    1)将75％木屑、20％麸皮、1％磷肥、1％石膏、3％腐殖土和水混合拌好，料水比为1:1.3，堆积发酵20天后，装料灭菌，接入羊肚菌菌种，封好袋口，置于22-25℃下培养至菌丝满袋，一段时间后脱袋；
    2)在腐殖土上铺放脱袋的羊肚菌菌棒，覆盖腐殖土，维持土壤含水量40-70％，控制培养温度6-18℃，光照每天4-12小时，培养；
    3)羊肚菌子实体成熟开始采收，收集腐殖土灭菌继续用于下一茬羊肚菌的培养；
    4)重复上述步骤5次，获得第一腐殖土。
  10. 一种低砷及重金属含量的冬虫夏草，其通过权利要求1-9任一项所述的方法繁育所得。
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