趙永昌 柴紅梅 陳衛(wèi)民 趙子悅 孔令艦

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羊肚菌子實體發(fā)育生物學(xué)（下）——生物學(xué)和非生物學(xué)因子對菌絲培養(yǎng)和子實體形成的影響

趙永昌 柴紅梅 陳衛(wèi)民 趙子悅 孔令艦

（云南省農(nóng)業(yè)科學(xué)院生物技術(shù)與種質(zhì)資源研究所，云南 昆明 650221）

3 羊肚菌發(fā)育生物學(xué)

羊肚菌的生活史是研究的難點，目前有提出兩種生活史[114, 115]，都認(rèn)為形成菌核和分生孢子是關(guān)鍵環(huán)節(jié)。儲存營養(yǎng)的菌核在子囊果發(fā)育過程中作用較大，既可以萌發(fā)為生殖菌絲，又可以直接產(chǎn)生子囊果。羊肚菌的菌核為假菌核，不同的可栽培出菇的菌株產(chǎn)菌核能力差別較大，甚至有菌種階段完全不產(chǎn)菌核的菌株也能很好地出菇；另一方面尚無菌核萌發(fā)成原基的直接證據(jù)。分生孢子的爭議則較大，主要是從自然基質(zhì)得到的分生孢子不能萌發(fā)，且羊肚菌產(chǎn)量與分生孢子產(chǎn)生量沒有直接相關(guān)性。使用覆膜技術(shù)種植羊肚菌有的分生孢子非常少但出菇不受影響，用圓葉楊種植也幾乎未發(fā)現(xiàn)有分生孢子形成。因此，羊肚菌的發(fā)育生物學(xué)基礎(chǔ)研究可能是解決羊肚菌穩(wěn)產(chǎn)、育種的關(guān)鍵，而實現(xiàn)室內(nèi)穩(wěn)定出菇則是研究的基礎(chǔ)。解決羊肚菌培養(yǎng)過程中遺傳的多變性是重中之重。

3.1 子囊孢子形成與萌發(fā)

子囊孢子的形成與萌發(fā)是羊肚菌發(fā)育的關(guān)鍵問題，羊肚菌子囊孢子具有多核現(xiàn)象，有的多達(dá)數(shù)十個核，這些核是同核體還是異核體尚不清楚。雖然有不少學(xué)者試圖對羊肚菌子囊孢子的形成進(jìn)行深入研究，但是由于未實現(xiàn)室內(nèi)栽培，難以找到不同發(fā)育時期的子囊孢子個體，導(dǎo)致研究困難。陳立佼借助梯棱羊肚菌的大田栽培，通過細(xì)胞核染色技術(shù)對子囊孢子的形成進(jìn)行研究，總結(jié)出子囊孢子形成的過程模式[116]，說明羊肚菌子囊孢子的多核是孢子形成后的有絲分裂導(dǎo)致的，多核子囊孢子應(yīng)是同核體，這方面的結(jié)果得到交配型基因鑒定證明。Chai等證明-20和子囊孢子群都是同核體，具有1-1和1-2交配型基因子囊孢子的比例與1?1無偏差，所以M-20和是異宗結(jié)合的[117]。有研究認(rèn)為栽培條件下子囊果形成孢子比較晚，自然生長的子囊果傾斜或倒伏狀態(tài)時才會形成孢子，此時子囊內(nèi)除孢子外基本呈透明狀態(tài)，不存在其他細(xì)胞器或大分子。也有不形成孢子的共生類型的羊肚菌菌株[118]。

在室內(nèi)條件下，羊肚菌的子囊孢子較易萌發(fā)，在濕度適宜時3小時就可見到芽管，因子囊孢子的多核性其可能從1-5端長出芽管萌發(fā)；在野外，影響子囊孢子萌發(fā)的因素很多，溫度、濕度、營養(yǎng)條件都可能影響。研究表明，子囊孢子萌發(fā)對溫度比較敏感，室內(nèi)溫度低至2 ℃都能萌發(fā)，但將孢子包埋在玻璃紙里放置在出菇點時，土壤溫度超過10 ℃都不萌發(fā)。當(dāng)土壤持續(xù)升溫到15 ℃時會抑制子囊孢子的萌發(fā)，地表的孢子一年后將失去活力。將經(jīng)滅菌的黑麥埋入地中，土壤溫度保持在10 ℃以下時，無論孢子還是菌核都容易在其上定植[119]。一定濃度范圍的MgSO4、KNO3、CaCl2、NaNO3對孢子萌發(fā)和菌絲生長有促進(jìn)作用[120]。子囊孢子萌發(fā)的菌絲一般仍為多核體細(xì)胞，同一子囊孢子萌發(fā)得到的初生菌絲不發(fā)生融合，也未觀察到不同子囊孢子萌發(fā)的菌絲之間發(fā)生融合[118, 121]。有研究表明，單孢菌株配對后能形成異核體，這與菌柄分離的菌株為異核體類似，間接表明其為異宗結(jié)合[114, 122, 123]。

3.2 多變的培養(yǎng)形態(tài)

羊肚菌的培養(yǎng)形態(tài)變異之大是目前大型真菌中少有的，相同培養(yǎng)條件下不同種或同種不同菌株在菌核形成的時間、形態(tài)、多少和大小等方面均有不同，在不同的培養(yǎng)條件下同一菌株菌核的產(chǎn)生和形態(tài)也不同，培養(yǎng)過程中的變化不僅類型多且重復(fù)性較差。雖然培養(yǎng)基組成、生長條件顯著影響羊肚菌生長速度[124～129]，但引起這些變化的原因是遺傳性的還是純粹生理性的尚不清楚。羊肚菌菌絲存在多核現(xiàn)象，這種多核仍舊是同核體。同核體菌株培養(yǎng)過程中變化穩(wěn)定，不存在由于核丟失而產(chǎn)生變化。

陳立佼等從單孢群體出發(fā)以產(chǎn)菌核能力為定性特征研究了培養(yǎng)特性變化[130]，單孢菌株按培養(yǎng)特征可分為9類（表3），同等條件下每一菌株的培養(yǎng)特性保持穩(wěn)定，在綜合馬鈴薯葡萄糖培養(yǎng)基（CPDA）、葡萄糖硝酸鈉瓊脂培養(yǎng)基（GN）、酵母膏胨葡萄糖瓊脂培養(yǎng)基（YPD）進(jìn)行轉(zhuǎn)接培養(yǎng)時，可成功地將產(chǎn)菌核菌株轉(zhuǎn)化為不產(chǎn)菌核菌株。同一子實體及不同子實體產(chǎn)菌核單孢菌株產(chǎn)核數(shù)量及分布，變化很大，對峙培養(yǎng)后單孢之間性狀會發(fā)生較大變化，包括菌核形態(tài)、菌絲形態(tài)、生長勢，特別是產(chǎn)菌核能力會消失和發(fā)生轉(zhuǎn)移：①同一子實體單孢菌株之間存在拮抗作用，對峙培養(yǎng)時相互接觸部位會形成交合線，出現(xiàn)濃密的生長區(qū)，將不產(chǎn)菌核的單孢菌株與同一子實體及不同子實體產(chǎn)菌核單孢菌株進(jìn)行對峙培養(yǎng)，即使來自相同子實體的單孢菌株之間也會出現(xiàn)拮抗作用。拮抗線兩側(cè)的菌絲和親本菌株相比形態(tài)和產(chǎn)核情況有很大變化，在各對峙組合中不同親本的組合拮抗線情況不同。②在對峙組合中由于拮抗作用，兩親本的生長勢不同，有拮抗的組合中產(chǎn)菌核菌株長勢明顯占優(yōu)，只是有的長勢處于弱勢，可能是由于產(chǎn)菌核菌株菌絲輻射狀快速生長，使其在平板上能迅速占據(jù)更多培養(yǎng)資源。③產(chǎn)菌核水平變化，拮抗作用對產(chǎn)菌核能力影響較大，有產(chǎn)核能力減弱的，有完全失去產(chǎn)菌核能力的，也有產(chǎn)菌核能力互換的（在組合中一個菌株將產(chǎn)菌核能力“轉(zhuǎn)染”，原來產(chǎn)菌核的菌株仍然產(chǎn)菌核，不產(chǎn)菌核的菌株能產(chǎn)菌核），可推測在對峙培養(yǎng)過程中與菌核形成相關(guān)的遺傳因子可在細(xì)胞間遷移，或是菌核產(chǎn)生與某種分泌物有關(guān)。該物質(zhì)有一定的趨向性，各菌株對其吸引能力的強弱決定了它的分布，從而影響了菌核的產(chǎn)生和分布。

表3 基于YPD上的培養(yǎng)特征的羊肚菌單孢菌株分組[130]

3.3 菌核形成與變化

3.3.1 純培養(yǎng)條件下菌核變化

菌核是一種由菌絲組織化形成的具有抵御逆境作用的致密菌絲結(jié)構(gòu)，逆境是菌核產(chǎn)生的重要基礎(chǔ)與條件；然而羊肚菌在營養(yǎng)豐富的環(huán)境中也能產(chǎn)生菌核。說明營養(yǎng)因素既可以刺激也可能抑制羊肚菌菌核的形成。盡管羊肚菌栽培取得了進(jìn)展，但仍具有挑戰(zhàn)性，因為即使按照專利也不能完全栽培出菇。多數(shù)研究認(rèn)為，要獲得栽培成功，必須獲得菌核，不少人曾經(jīng)將精力集中在菌核產(chǎn)生研究上。對于羊肚菌菌核的形態(tài)和形成過程有不同看法，Alvarado-Castillo等[131]的分類比較合理，包括七個階段：①菌絲生長，②初級菌絲生長和分枝，③次級菌絲纏繞，④大量菌絲聚集，⑤菌絲聚合生長，⑥菌核形成，⑦菌核生長與成熟，成熟伴隨厚垣孢子和分生孢子形成。

影響菌核形成的因素較多，包括環(huán)境因子和營養(yǎng)因子[71, 132]，營養(yǎng)因子涉及種類和濃度。在不同蔗糖濃度的培養(yǎng)基上，菌絲生長速度、菌核顏色差別較大，且形成菌核的菌絲體外表會有一些具備儲蓄能力的氣泡[133, 134]。氮源的影響高于碳源[135]，在含核糖、半乳糖、山梨糖和甘露醇的培養(yǎng)基上較難形成菌核，而NaNO3和酵母提取物有利于分散型菌核形成[136, 137]。復(fù)合營養(yǎng)物如玉米、小麥、燕麥和黑麥等，對菌核的形成有促進(jìn)作用[138]。而饑餓可以誘導(dǎo)和增加假菌核的形成，假菌核中丙二醛含量增加與脂肪的過氧化和氧化壓力有關(guān)[139]。影響菌核形成的非營養(yǎng)因素包括光照、溫度、有機酸、環(huán)境腐敗產(chǎn)物的積累、酚類化合物、多酚氧化酶活性、機械屏障、疏基修飾因子，以及滲透壓等，能增加或減弱氧化應(yīng)激的生長因子可能都有促進(jìn)或抑制菌核分化的作用。

3.3.2 菌核生理變化

木質(zhì)素降解酶是大型真菌最重要的酶之一，其參與人工栽培的子實體形成。在羊肚菌生活史中，菌核是羊肚菌從菌絲到子實體的中間態(tài)。研究表明，在不同培養(yǎng)基上，羊肚菌菌核形成過程中木質(zhì)素降解酶的活性有較大變化，不同培養(yǎng)基上都產(chǎn)生漆酶（LAC）、木質(zhì)素過氧化物酶（LiP）和錳過氧化物酶（MnP）等木質(zhì)素降解酶，在麥粒培養(yǎng)基上LAC活性最高，而在谷殼培養(yǎng)基上則是MnP和LiP最高；在土壤復(fù)合培養(yǎng)基上，從菌核形成到成熟都能產(chǎn)生漆酶，都能測到漆酶活性[140, 141]。由于基質(zhì)對菌核形成及相關(guān)酶分泌有誘導(dǎo)作用，所以羊肚菌栽培過程中栽培種配方的選擇對菌核形成及后期胞外酶的表達(dá)有決定性作用。

3.3.3 菌核形成過程中基因表達(dá)變化

羊肚菌菌核形成能力的差異，表面上受環(huán)境因素影響，實際上是由菌株本身的遺傳多樣性決定的。Chen等[142]利用mRNA差異顯示技術(shù)對同一子實體分離的在相同培養(yǎng)條件下得到的產(chǎn)菌核和不產(chǎn)菌核的羊肚菌單孢進(jìn)行研究，得到了陽性的假設(shè)蛋白及未知的差異片段58個，其中26條片段為已知蛋白功能片段、9條與已知物種的假設(shè)蛋白編碼片段相似，23條為未找到假設(shè)蛋白片段。這些片段對應(yīng)的基因可能控制著羊肚菌產(chǎn)菌核與否或能力高低，為在遺傳和基因調(diào)控水平研究羊肚菌產(chǎn)菌核的機理提供了重要的遺傳信息。在確定功能的基因中，氨基丁酸通透酶片段、外膜家族蛋白相關(guān)蛋白片段和人類角蛋白結(jié)合蛋白片段都與羊肚菌逆境應(yīng)激和結(jié)構(gòu)適應(yīng)性變化有關(guān)，即得到的差異片段與植物中的脂質(zhì)代謝、氮代謝、逆境應(yīng)激及細(xì)胞周期調(diào)控有關(guān)。這些片段可能是羊肚菌的氧化應(yīng)激反應(yīng)的上游誘導(dǎo)因子或下游產(chǎn)物，需要得到片段全長并進(jìn)行更深入的功能研究才能確定。表明羊肚菌菌核形成能力可能更多地與菌株對外界環(huán)境適應(yīng)性的遺傳多樣性有關(guān)。

3.3.4 菌核與子實體形成能力之間的關(guān)系

雖然最早的研究認(rèn)為，菌核是羊肚菌生活史中最重要的階段[9]，易出菇的火燒地在兩年內(nèi)觀測到的羊肚菌菌核比非火燒地多得多[143]。但對羊肚菌的生活環(huán)境調(diào)查研究發(fā)現(xiàn)，菌核并不是所有種的羊肚菌生活史的必須階段，不同種在生活史的進(jìn)化上有區(qū)別[71]。目前實驗證明，菌核的有無與出菇與否及產(chǎn)量高低之間沒有必然的聯(lián)系，有菌核產(chǎn)生的菌株不一定出菇，很少形成菌核的菌株同樣也可以出菇[144]。雖然在栽培實踐中，多數(shù)種植者認(rèn)為需要栽培種長滿菌袋（瓶）并形成菌核后才可以播種，但不少菌絲長滿后就播種的出菇時間縮短，且產(chǎn)量穩(wěn)定。

3.4 分生孢子

羊肚菌栽培過程中會形成分生孢子，Ower[9]也有提及。分生孢子在目前假設(shè)的羊肚菌生活史中是重要的階段。研究表明，羊肚菌分生孢子多數(shù)為單核[145]，且產(chǎn)生與否與出菇與否無直接關(guān)系[144]。但也有認(rèn)為分生孢子類似于植物花粉的作用，對出菇有重要的意義[146]。

圖2 羊肚菌產(chǎn)生分生孢子（左）與不產(chǎn)生分生孢子（右）

明確羊肚菌分生孢子的生物學(xué)功能，首先須解決無菌培養(yǎng)條件下分生孢子的形成和分生孢子的萌發(fā)兩個難題。分生孢子可能是一種休眠體，其萌發(fā)需要特定的環(huán)境條件，目前尚無在栽培過程中觀察到分生孢子萌發(fā)的報道。根據(jù)我們研究，在滅菌基質(zhì)上生長的羊肚菌可以形成分生孢子（圖2）：①分生孢子形成與器皿無關(guān)，不同的器皿培養(yǎng)都能形成分生孢子；②分生孢子形成與接種量密切相關(guān)，當(dāng)接種量大時不形成分生孢子；③分生孢子形成與溫度相關(guān)，溫度超過24 ℃，形成的分生孢子較少，而10～24 ℃間的差別不明顯，但形成時間有差異；④在最優(yōu)條件下，不同菌株分生孢子的產(chǎn)生量差別較大，同一物種分生孢子形成數(shù)量多孢菌株>單孢菌株>組織分離菌株；⑤分生孢子形成與土壤基質(zhì)的顆粒度密切相關(guān)，顆粒度小分生孢子數(shù)量少。

A：分散型原基；B：叢生型原基；C：分散性菌核周邊的原基；D：大菌核周邊的原基。

3.5 原基形成分化與子囊果形成

Ower[9]首次較為完整地描述了羊肚菌子實體，之后不斷有人對羊肚菌發(fā)育過程進(jìn)行描述[69,70,144]，基本特征是在基質(zhì)表面上從一個點逐步形成菌絲團(tuán)，菌絲團(tuán)上下組織化分化出菌蓋和菌柄，菌柄往下生長吸收營養(yǎng)，菌蓋往上發(fā)育逐步成熟。與其他菇類原基下面是密集的菌絲不同，大田中經(jīng)常觀察到羊肚菌原基是由一個小點形成的，羊肚菌子實體發(fā)育的營養(yǎng)是如何供給輸送的尚不清楚。我們研究發(fā)現(xiàn)光溫濕可控條件下原基可以形成（圖3）：①單一交配型的菌株都不能形成原基，不同物種多孢分離菌株和組織分離菌株在原基形成的形態(tài)和數(shù)量上有差異，但無規(guī)律；②原基形成的數(shù)量與分生孢子形成相關(guān)，沒有分生孢子（包括接種量大、24 ℃培養(yǎng)）的都未形成原基；③雖然很難看到原基由菌核分化而來，但菌核多的原基數(shù)量多且密。雖然是初步結(jié)果，且在原基的分化和幼菇發(fā)育控制上尚需進(jìn)一步研究，但結(jié)合大田的種植結(jié)果可以為羊肚菌優(yōu)良菌株鑒定體系的建立奠定基礎(chǔ)。

圖4 羊肚菌栽培兩種不同的出菇方式

3.6 出菇方式與富營養(yǎng)栽培

大田栽培羊肚菌出菇方式有見原基和不見原基兩種（圖4），有以下特點：①與菌株特性有關(guān)，多數(shù)菌株是見原基出菇，有的則易不見原基出菇，也有兩種出菇形式都常見；②與土壤的酸堿度、顆粒大小、濕度等相關(guān)；③與栽培方式如覆土厚度相關(guān)；④不見原基出菇的成菇率高，但其發(fā)生率更低；⑤同一菌株同一田塊兩種出菇方式都會發(fā)生；⑥不見原基出菇往往伴隨假根的形成（圖5）。

圖5 不見原基出菇形成的假根

圖6 營養(yǎng)袋內(nèi)形成的原基

兩種出菇方式的機理尚不明確，對其研究有利于提高成菇率，可控制出菇的密度和整齊度。實現(xiàn)對不見原基出菇方式的控制更有利于實現(xiàn)工廠化栽培。不見原基出菇形成的假根應(yīng)該是環(huán)境因素引起而不是物種的特點，不應(yīng)作為分類依據(jù)。沙質(zhì)土壤滴灌或噴灌易導(dǎo)致土壤微移動，這種土壤移動是“見原基出菇”方式中原基因移動而懸空死亡的重要原因，提高成菇率可在催菇后進(jìn)行噴霧保濕。

工廠化栽培的難點是通過富營養(yǎng)栽培提高單位面積的產(chǎn)量。但富營養(yǎng)情況下較難出菇，在栽培中曾發(fā)現(xiàn)營養(yǎng)袋內(nèi)形成大量原基（圖6），提示條件適宜的情況下富營養(yǎng)栽培是可行的。

致謝 本文圖5、圖6由四川省平昌縣金源生物科技有限公司孫勝先生提供，特此感謝。

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