Guanglu GONG, Tongjing YANG, Qin WANG, Yang GUI, Wanbing HUANG, Yingying LU, Hongyu LIU, Xiaorun HUANG, Guosheng ZHU*

1. Institute of Crop Variety Resources, Guizhou Academy of Agricultural Sciences/Guizhou Provincial Key Laboratory of Edible Fungus Breeding/Modern Agricultural Technology System of Edible Fungi in Guizhou Province, Guiyang 550006, China; 2. Guizhou General Station of Crop Technology Extension, Guiyang 550001, China

Abstract China is the earliest country to eat Dictyophora and to realize its artificial domestication. At present, 12 species of Dictyophora have been reported worldwide, 7 species of which are found in China. D. rubrovolvata, D. echinovolvata and D. indusiata have been cultivated on a large scale, and the main producing areas are Zhijin in Guizhou Province, Shunchang and Jiangle in Fujian Province, and Qingchuan and Changning in Sichuan Province. The cultivation of D. rubrovolvata had experienced 4 stages: wild tending, casserole cultivation, outdoor simple greenhouse cultivation and rapid development of new cultivation techniques. The present integrated cultivation technique of bag removing and soil covering of D. rubrovolvata were introduced in detail from the aspects of production and selection of high-quality fungi bags, bag removing and soil covering, spawn running, as well as fruiting management and harvesting. As one of the "ten main promoting technologies" of Guizhou Province in 2022, it had realized rapid propagation of liquid strains and supporting cultivation of improved varieties and methods. The suitable strains were selected to support understory cultivation, layer cultivation, basket cultivation, factory cultivation and other modes, which had short cultivation period and can realize annual production and supply. With the development of the industry, it is expected that low-cost secondary fermentation or tertiary fermentation cultivation technology will be more widely used.

Key words Dictyophora industry, Dictyophora rubrovolvata, Dictyophora echinovolvata, Dictyophora indusiata, Fungi bag removing, Soil covering cultivation

1 Introduction

Dictyophora, also known as bamboo fungus, belongs toDictyophora, Phallaceae, Phallales, Gasteromycetes, Basidiomycotina, Eumycota[1]. Due to rich nutrition[2]and beautiful appearance, it enjoys the reputations of "modern health food", "pinnacle of human plant food", "flower of the fungi", "queen of the fungi", "flower of delicacies from mountains",etc.[3].

China is the earliest country to eatDictyophoraand to realize its artificial domestication. There were records aboutDictyophorainMiscellaneousMorselsfromYouyangwritten by Duan Chengshi in the Tang Dynasty. A total of 12 species ofDictyophorahave been reported worldwide, mainly distributed in China, India, Sri Lanka, Africa, Australia, North America, South America and the East West Indies. There are 7 species ofDictyophorain China, includingD.indusiata,D.duplicate,D.multicolor,D.cinabarina,D.merulina,D.rubrovolvataandD.echinovolvata[4], andD.rubrovolvataandD.echinovolvatawere discovered and named by Chinese scholars. The wild resources ofDictyophorain China are mainly distributed in Yunnan, Guizhou and Sichuan.

2 Present situation of Dictyophora industry in China

China has now realized large-scale planting ofD.rubrovolvata,D.echinovolvataandD.indusiata, and the producing areas are distributed in Sichuan, Fujian, Guizhou, Jiangxi, Yunnan, Guangxi and Hunan. According to statistics from the China Edible Fungi Association, the national production ofDictyophorareached 59 300 t in 2012, and had exceeded 60 000 t by 2021, indicating thatDictyophoraindustry is growing.

D.rubrovolvatain Guizhou,D.echinovolvatain Sichuan andD.indusiatain Fujian have been developing in clusters as leading varieties, forming a "tripartite" trend of development inDictyophoraindustry. Zhijin County of Guizhou Province was honored as "the hometown ofDictyophorain China" by the China Edible Fungi Association in August 2000, and was certified as "ZhijinDictyophora" national geographical indication product in September 2010. In May 2008, Shunchang County of Fujian Province was awarded the title of "the hometown ofDictyophorain China" by the China Edible Fungi Association, and was certified as "ShunchangDictyophora" and "JiangleDictyophora" national geographical indication products in November 2009 and February 2015, respectively. In December 2012 and December 2015, Qingchuan County and Changning County of Sichuan Province were certified as "QingchuanDictyophora" and "ChangningDictyophora" geographical indication protection products, respectively. In August 2014, Changning County in Sichuan Province was honored as "the hometown ofDictyophorain China".D.rubrovolvatais one of the most expensive varieties ofDictyophorabecause of its fleshy texture, durability after cooking for a long time, crisp and tender taste of fresh and dry food, and high edible and health value.

3 Development history and present situation of D. rubrovolvata industry

In 1972, US President Richard Nixon sent special envoy Henry Kissinger to Beijing, where he was feasted by Premier Zhou Enlai, and thus "chu-sheng egg & vegetable soup" became famous at home and abroad[5]. In the meantime, China raised the upsurge of artificial cultivation study ofD.rubrovolvata. ZhijinD.rubrovolvatawas successively awarded the gold medal at the 5thChina New Technology and New Products Exhibition in 1993, the gold medal at the China High Quality Agricultural Products and Scientific and Technological Achievements Exhibition in 1993, and the gold medal of Chengdu National Spark Boutique in 1994. In 2005, Sanjiazhai Village of Houzhai Township obtained the "certificate of organic products"; in 2007, it was awarded as "famous, special and excellent agricultural products" in the 3rdGuizhou Agricultural Products Exhibition, and "gold award" and "quality product award" in the 2007 China (Changsha) International Edible Fungus Industry Exhibition; in 2010, it was certified as a national geographic indication product; in 2014, it was awarded as China’s famous agricultural products by the Ministry of Agriculture; on 27 July 2020, ZhijinD.rubrovolvatawas included in theProtectionListofEU-ChinaGeographicalIndicationsAgreement(the second batch); in September 2021, it was included in the second batch of national famous, special and excellent new agricultural products list by the Agricultural Product Quality and Safety Center of the Ministry of Agriculture and Rural Affairs. During the poverty alleviation campaign,D.rubrovolvatabecame the star industry in Guizhou.

The industry ofD.rubrovolvatahas gone out of Zhijin, Guizhou and developed in the whole province and even the whole country. During the national planting peak in 2020, the total scale ofD.rubrovolvataexceeded more than 4 000 ha, about 3 666.7 ha of which were distributed in Guizhou and about 1 000 ha in Zhijin County. In 2021, the national planting scale was about 2 000 ha, about 1 333.3 ha of which were distributed in Guizhou and about 333.3 ha in Zhijin County. At present, the whole industrial chain ofD.rubrovolvatahas been basically formed in Henan and Anhui, and it is also cultivated in some areas of Yunnan, Sichuan, Guangxi, Guangdong, Fujian, Zhejiang and Hainan.D.rubrovolvatahas gradually emerged as a popular product in front of the world, becoming a high-quality variety leading the nationalDictyophoraindustry.

4 Development history of the cultivation technique of D. rubrovolvata

4.1 Wild tending stageWild tending technology mainly involves harvesting wild mycelia andDictyophoraeggs, and transplanting them with the soil as a whole in bamboo forests close to the planter and easy to manage, which is the product of the development of wild foraging tide (Fig.1). In the 1960s and 1970s, foraging wild fungi was prevailing. In order to avoid being picked, the mature eggs ofD.rubrovolvatawere transplanted to home or in a bamboo forest or indoor area close to home, and harvested after concentrated fruiting, thus forming a model of understory transplanting, especially typical in Zhijin County. In Zhijin County, wildD.rubrovolvatacan be found in almost every bamboo forest, which is related to the operation[6].

4.2 Indoor casserole cultivation stageIn 1972, the "chu-sheng egg & vegetable soup" hosted by Premier Zhou Enlai to Henry Kissinger, the special envoy of the United States, made GuizhouDictyophorafamous all over the world. It was in great demand immediately, and the price once had reached 4 000 HKD/kg. Wild resources were in short supply. In this context, indoor casserole cultivation technology was gradually formed[7](Fig.2). Bamboo pieces or wood blocks were used to artificially cultivate the fungi which were then covered with humus soil dug from the mountains. As plastic baskets and pots were scarce at that time, each household cultivated the fungi with a casserole used for cooking or livestock grain. Because of water and heat preservation and ventilation, casserole is very suitable for the growth ofD.rubrovolvata, with higher yield. The cultivation mode madeD.rubrovolvataindustry become one of the main industries in Zhijin County. At that time, it was even popular among Zhijin people that "It is better to plant a single casserole ofDictyophorathan to receive a year of national grain." Many people quit their jobs and plantedDictyophorain casserole at home. The promotion and development of this technology had driven the casserole industry in Zhijin County and its surrounding areas, and also formed a casserole street in Zhijin County.

4.3 Outdoor simple greenhouse cultivation stageThe domestication and cultivation of wildDictyophorawas explored in 1983. In 1986, the artificial outdoor cultivation ofD.rubrovolvatawas successful. In the early 1990s, 32 townships (towns and sub-district offices) in Zhijin County plantedDictyophora. More than 30% of the farmers in Zhijin County had mastered the traditional cultivation techniques ofDictyophora. At its peak, the planting area ofDictyophorain Zhijin County was nearly 666.7 ha. The core technologies are single-step separation of strains, production technology of saline bottle strains, and popularization and application of wood block cultivation technology(Fig.3).Dictyophoraeggs are collected and directly separated into 500 mL saline glass bottles to produce strains;Betulaluminferais the dominant material for cultivation; the logs are cut into wood pieces (20-30 cm long, 5-8 cm wide, 2-5 cm thick), or crushed into 3-5 cm wood pieces, boiled with water for about 1 h for complete absorption of water and sterilization; humus soil is used as the covering soil, and pine needles are the main coverings; the framework of the greenhouse is mainly built of bamboo and wood, with a height of about 2 m, and covered with thatch or straw curtains around and at the top[8].

This stage is the main development stage ofD.rubrovolvata. Due to the continuous cropping obstacles, the planting base has to be replaced every year. Over the past 30 years,D.rubrovolvatahas formed a development mode of supplying strains in Zhijin, planting in the whole province, and selling back to Zhijin.

4.4 Fungi bag removingSince the 21stcentury, agricultural and forestry wastes such as straw and wood chips have replaced wood to cultivate edible fungi, and become a new direction of industrial development[9]. The edible fungi team of the Institute of Crop Germplasm Resources, Guizhou Academy of Agricultural Sciences began to conduct relevant research in 2009, and started the research of new cultivation technology in 2011. In 2012, the Science and Technology Department of Guizhou Province established a special project on the cultivation technology of substitute fermentation, and carried out the cultivation research of straw fermentation in Zhijin in that year. In 2013, the pilot test was carried out in the Guizhou Academy of Agricultural Sciences. In 2014, the cultivation technology of loose sand sawdust fermentation material was studied in Taijiang County. From 2015 to 2016, "loose sand sawdust + straw + miscellaneous wood chips" was successful in the pilot demonstration. After years of ripening, the "cultivation technique of bag removing and soil covering ofD.rubrovolvata" was selected as one of the top ten agricultural technologies in Guizhou Province in 2022. The technology achieved the industrial upgrading goals of high quality, high yield and high efficiency, improved the overall level of cultivation technology, and further promoted the rapid development of related industries.

There are many application modes for cultivation technique of bag removing and soil covering ofD.rubrovolvata(Figs.4-6), with strong practicability. The production cycle ofD.rubrovolvatastrains is shortened from 18 months to about 6 months by using liquid strains to achieve rapid propagation. Bag removing and soil covering ofD.rubrovolvatais featured by easy operation, easy fruiting, less diseases and insect pests, and shortened cultivation period, and can be developed in multiple modes such as understory cultivation, layer cultivation, basket cultivation and factory cultivation. The cultivation technique expands the scope of adaptation ofD.rubrovolvataand can be popularized throughout the country. The selection of different suitable varieties can realize the cultivation of good varieties and methods. For example, "Qianyou 1" is suitable for understory or simple greenhouse planting; "Qianfeng 1" is suitable for factory and basket cultivation; "yzs020", because of wide adaptability to temperature, is suitable for layer cultivation and can be cultivated with straw and other materials rich in cellulose.

5 Cultivation points of bag removing and soil covering of D. rubrovolvata

5.1 Production and selection of high quality fungi bagsThe matrix material is composed of 75% mixed sawdust, 5% cottonseed shell, 17% wheat bran, 0.7% brown sugar, 1% lime, 1% gypsum, 0.2% potassium dihydrogen phosphate, and 0.1% magnesium sulfate. Polythene bags with folded corners (37 cm long, 16 cm wide, 0.06 mm thick) are used, with a loading capacity of about 1 100 g. After loading, the rings are set and the breathable cap is covered. After sterilized at 121 ℃, 0.1-0.13 Mpa for 120 min and cooled to 25 ℃, the liquid strain is inoculated at the volume of 30-40 mL/bag. The fungi bag will be fully covered as cultured at 22-23 ℃ in the dark for 70-80 d.

High quality fungi bag is characterized by short age, no pollution, no atrophy, no fungi burning at high temperature, and can be identified by the way of "observing, smelling, asking and cutting".

The first step is asking, including the raw material of the formula, the form of the fungus, the inoculation time, the culture method, the storage temperature and the transportation method. The main raw material should be hard wood, with the proportion of fine and auxiliary materials not less than 20%; liquid strains should be inoculated in industrial culture, but not in other cultivation modes; the fungal age should not exceed 120 d; the suitable incubation temperature is 22-24 ℃, and the storage temperature is 4 ℃; during transportation, the interior temperature of the carriage shall not exceed 28 ℃, and the fungi bag shall not be squeezed, rained or exposed to the sun. The second step is observing: the fungi bag with white and dense mycelia, no atrophy, no yellow water, and no extrusion deformation should be selected. The third step is cutting: several fungal bags are randomly selected and touched to feel the degree of softness and hardness, and fungi bags with strong hardness are selected. The forth step is smelling: smell when unpacking the fungi bag and listen to the sound when forcing apart the fungi bag with the hands. The fungi bag of high quality has the fragrance of hypha ofDictyophora, without musty, sour odor and other foreign flavors, and the sound is clear and melodious when forcing apart the fungi bag.

5.2 Bag removing and soil coveringThe fungi bags are removed and placed side by side with a spacing of 5-10 cm (5 cm for industrial cultivation and 10 cm for other cultivation modes) on the furrow surface, and then covered with soil. The thickness of soil cover should be adjusted flexibly according to different cultivation modes. The thickness of soil cover is 3 cm for factory cultivation, 3-5 cm for simple greenhouse cultivation, basket cultivation and layer cultivation, and about 10 cm for understory cultivation. Finally, the turtleback shaped furrow surface can be formed.

5.3 Management of spawn running and fruitingAccording to the biological characteristics ofD.rubrovolvata, the following 5 elements are followed for the management of spawn running and fruiting.

(i) Moisture. The moisture content of fungi bag is about 60%; the soil moisture content is 20%-22%, and the soil interstitial moisture is 60%-70%. The soil surface moisture should be 60%-70% in the stage of spawn running and 70%-80% in the stage of fruiting; the relative air humidity should be 70%-80% in the stage of spawn running and 85%-95% in the stage of fruiting. (ii) Fertilization. The fungi bag is mainly composed of hard wood, with about 20% refined and auxiliary materials; when the primordium grows to a diameter of more than 2 cm, nutrient solution can be added (brown sugar 0.05%-0.10%, amino acid 0.01%-0.05%, zinc sulfate 4‰, monopotassium phosphate 4‰, triacontanol 0.5 mg/L). (iii) CO2concentration. The volume concentration of CO2in the interstices of soil should be not more than 0.1%, and the volume concentration of CO2in the air should be not more than 0.06%. (iv) Temperature. The temperature for the growth ranges from 5 to 30 ℃, and the optimum temperature is 22-26 ℃. The soil will not freeze under the ground layer greater than 1 cm. The temperature for fruiting stage ranges from 16 to 28 ℃, and the optimum is 20-24 ℃. The temperature should be not more than 28 ℃ within 20 cm above the ground during the fruiting stage. (v) Light. Light is not required in the growth stage of mycelia, but scattered light is needed in the fruiting stage, with the light intensity of 200-500 lx.

Fig.1 Fruiting process of wild Dictyophora rubrovolvata (shot in Fuzhou, Jiangxi in 2008)

Fig.2 Cultivation of Dictyophora rubrovolvata in casserole (shot by Dai Jihua in the 1980s)

Fig.3 Cultivation of Dictyophora rubrovolvata outdoors in simple greenhouses

Fig.4 Understory cultivation (left) and basket cultivation (right) of Dictyophora rubrovolvata

Fig.5 Simple greenhouse planting (left) and layer cultivation (right) of Dictyophora rubrovolvata

Fig.6 Factory cultivation of Dictyophora rubrovolvata

5.4 HarvestingFor understory cultivation, it is recommended to collect fungi balls (commonly known asDictyophoraeggs) for concentrate fruiting, while for other cultivation modes, it is recommended to collectDictyophoraduring veiling period (commonly known asDictyophoraflowers).Dictyophoraflower harvesting: pick at the beginning of the emergence of fruiting body or when the fruiting body has been scattered.Dictyophoraflowers are generally harvested in the morning in the following steps: uncovering →root cutting→volva removing(Fig.7), keeping the appearance clean and the shape intact.Dictyophoraegg collection: the matureDictyophoraeggs are selected and held in one hand, and the eggs are cut off from the base with a knife in the other hand, trying not to destroy the surrounding mycelia and smallDictyophoraeggs. The collectedDictyophoraeggs should be placed in the fruiting room in time for concentrated fruiting (Fig.8). The immatureDictyophoraeggs that are collected unintentionally will not proceed to fruiting.

5.5 ProcessingDictyophoraflowers should be dried within 2 h after harvesting, which are dried at 55 ℃ for 4 h and 45 ℃ for 6 h.Dictyophoracovers are soaked in warm water at 60-70 ℃ for 4-6 h, then rinsed with water, and dried every 3-5 sets together, or can be set back onDictyophoraflowers to dry together. The cover is fragrant and odorless, with high nutritional value, and can also be sold as a commodity.

Fig.7 Uncovering (left), root cutting (middle) and volva removing (right) of Dictyophora rubrovolvata

For defective products, the indusium can be processed separately intoDictyophorayarn; the stipe can be cut into small segments of about 5 cm and processed intoDictyophorasegments (Fig.9).

As for processing of smallDictyophoraeggs, the immature smallDictyophoraeggs that are collected unintentionally can not break the shell normally, or the flowers are too small to be sold properly. Therefore, they can be directly sliced and dried intoDictyophorapieces. As for iron egg processing, the loss of water inDictyophoraeggs will lead to the inability to produce mushrooms. TheDictyophoraeggs (iron eggs) that can not break the shell after 3 d of concentrated fruiting will no longer break the shell and produce mushrooms later, but will decay with time. Therefore, they must be processed intoDictyophoraeggs,Dictyophorafetuses andDictyophoraplacentas in time.

Fig.8 Concentrated fruiting of Dictyophora rubrovolvata

Fig.9 Types of Dictyophora products

5.6 Integrated pest managementThe main insect pests ofD.rubrovolvatainclude mites, springtails (short-tailed springtails), ants, nematodes, slugs, gold moths and so on. The main diseases are yellow stunt, brown rot, soft rot, viral disease,Neurosporacrassa,Aspergillus, green mold,Trichoderma,Mucor, slime mold (Stemonitisfusca,etc.), cap fungi,Coprinus,etc.The diseases and pests are controlled following the principle of "prevention first, comprehensive control", while agricultural and physical control are the major means, supplemented with chemical control.

Agricultural control: reasonably arrange the production season according to the local climatic conditions and variety characteristics; control the quality of raw materials and standardize the preparation of fungi bags; ensure the cultivation management in place; and ensure the environmental hygiene of raw material warehouses, stacking sites and cultivation sites.

Physical control: colored sticky trap, sweet food, field traps and trap lights are used to trap insect pests; air filtration, ultraviolet light, ozone and other devices are used for disinfection.

Chemical control: pesticides registered in the production of edible fungi should be used in strict accordance with the provisions of GB/T 8321 (all parts). The use of pesticides is prohibited within 15 d before harvest.

6 Prospect and forecast

With the breakthrough of new cultivation technology, the fleshy, fragrant and nutrient-richD.rubrovolvata, a kind of rare edible fungi, will undoubtedly set off a nationwide planting boom, and the development scale will show a negative relationship with sale price.

D.rubrovolvatawill certainly become the first choice of fresh edibleDictyophoraproducts, with the trend of annual production and supply, and will also become the preferred variety to develop the understory economy.

According to the cultivation history ofD.rubrovolvata, its cultivation mode will be dominated by factory annual production mode with rapid breeding and fermentation technology of liquid strains, and intelligent regulation as the core technologies.