In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora

Amanita caesareoides is a sister species of Amanita caesarea, also known as Caesar’s mushroom and one of the most desirable edible mycorrhizal mushrooms. However, cultivation of Caesar’s mushrooms has not yet been successful due to the difficulties involved in establishing pure cultures. In this study, we established pure cultures of four Asian Caesar’s mushroom species, i.e., A. caesareoides, Amanita javanica, Amanita esculenta, and Amanita similis, which were identified by sequence analysis of their rDNA internal transcribed spacer (ITS) region. Five selected isolates in A. caesareoides, A. javanica, and A. esculenta were tested for ectomycorrhizal syntheses with axenic Pinus densiflora seedlings in vitro. Ectomycorrhizal tips of each fungal isolate tested were observed on pine lateral roots within 5 months of inoculation. Seventeen pine seedlings that formed ectomycorrhizas in vitro with these three Amanita species were acclimatized under non-sterile conditions. Seven months following acclimatization, ectomycorrhizal colonization by A. caesareoides was observed on newly grown root tips, which was confirmed by polymerase chain reaction restriction fragment length polymorphism analysis of the fungal rDNA ITS region. Two other Amanita species also survived during ectomycorrhizal acclimatization. These results suggest that the cultivation of A. caesareoides and its relatives can be attempted through mycorrhizal synthesis using P. densiflora as a host. This is the first report of in vitro mycorrhization of Asian Caesar’s mushrooms and their acclimatization under non-sterile conditions.     

Comparisons of ectomycorrhizae on pinyon pines (Pinus edulis; Pinaceae) across extremes of soil type and herbivory

Using field and greenhouse studies, we examined the relationships among pinyon pines (Pinus edulis), their ectomycorrhizal mutualists, and their moth herbivores as a function of soil fertility. We studied two soil types—the ash and cinder soils of the San Francisco volcanic field and nearby sandy loam soils. In the field, pinyons growing in cinders suffered from reduced moisture, negative nitrogen mineralization rates, low phosphate levels, reduced growth, and high moth herbivory relative to pinyons growing in sandy loam. Pinyons growing in cinders also had twofold higher levels of ectomycorrhizal colonization than their noncinder counterparts. Similarly, in the greenhouse, seedlings grown in cinders had higher levels of ectomycorrhizal colonization and greater numbers of ectomycorrhizae than seedlings grown in sandy loam. Seedling shoot growth was significantly enhanced by ectomycorrhizae in both soils. These patterns support three conclusions. First, field and greenhouse studies demonstrated that trees growing in nutrient-poor soils had higher levels of ectomycorrhizal colonization than trees growing in better soils. Second, across soil types, variation in ectomycorrhizal colonization was better predicted by soil fertility than by herbivory. However, herbivory negatively affected ectomycorrhizae in the stressful cinder environment. Third, although mycorrhizae can be parasitic under some conditions, ectomycorrhizae had mutualistic impacts on pinyon seedlings across the environmental extremes we studied.     

An ectomycorrhizal fungus,Cenococcum geophilum,in a coastal pine forest has a high tolerance for an insecticide used to control pine wilt disease

Cenococcum geophilum Fr., one of several ectomycorrhizal species associated with black pine (Pinus thunbergii Parl.), is dominant in the coastal forests of Japan, even under adverse abiotic environmental conditions. In these forests, many tonnes of Sumipine® (fenitrothion) are applied every year to protect P. thunbergii from pine wilt disease, which is transmitted by a beetle. Here, we examined the effect of this insecticide on the species of fungi found as ectomycorrhizae on naturally regenerated P. thunbergii seedlings collected from coastal forest sites that had or had not been sprayed with fenitrothion. The proportion of C. geophilum ectomycorrhizae on black pine root tips was significantly higher in areas where fenitrothion had been applied than in areas where it had not. We measured the in vitro mycelial growth of C. geophilum as well as other ectomycorrhizal fungi of coastal black pine, Rhizopogon roseolus (Corda) Th. Fr. and Pisolithus arhizus (Scop.) Rauschert, at three levels of fenitrothion (density: 1.32 g/cm³), i.e., 0, 0.1 and 0.2 mL L^?1. The growth of all three species decreased significantly as the fenitrothion dosage increased. However, the reduction of mycelial growth in response to fenitrothion was lower in C. geophilum than in the other two species. These results suggest that C. geophilum has a high tolerance for fenitrothion, which may explain its dominance over other ectomycorrhizal species in coastal forests in Japan where fenitrothion is routinely sprayed.

     

Mycorrhization between <Emphasis Type="Italic">Cistus ladanifer</Emphasis> L. and <Emphasis Type="Italic">Boletus edulis</Emphasis> Bull is enhanced by the mycorrhiza helper bacteria <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Migula

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.     

Habitat matters: The role of spore bank fungi in early seedling establishment of Florida slash pines

This study investigated broad patterns in communities of ectomycorrhizal fungi from three Florida habitats (sandhills, scrub, and pine rocklands) and the ability of spore bank fungi to associate with Pinus elliottii (slash pine) and Pinus densa (south Florida slash pine). Efforts to replant pines in the endangered pine rocklands are vital to the persistence of this habitat, yet little is known about the ectomycorrhizal fungi communities or how they may differ from those in other pine-dominated habitats in Florida. We used high-throughput amplicon sequencing (HTS) to assess baseline fungal communities and greenhouse bioassays to bait ectomycorrhizal fungi using seedlings. HTS soil data recovered 188 ectomycorrhizal species but only a few subsequently colonized the bioassay seedlings. We recovered 21 ectomycorrhizal species on pine seedlings including common spore bank fungi such as Cenococcum, Suillus, and Tuber, but Rhizopogon species were dominant across all sites and habitats. Habitat type and site were significant variables influencing the community composition of the total soil fungal community, soil ectomycorrhizal community, and the fungi found on seedling root tips. However, we found no significant differences between the ectomycorrhizal communities on seedling roots from the two Pinus species.     

Ectomycorrhizae and ectomycorrhizal fungal fruit bodies in pine stands differentially damaged by pine wilt disease

We surveyed ectomycorrhizae, ectomycorrhizal fungal fruit bodies, and soil physical properties in one heavily damaged and two lightly damaged pine stands on Mt. Tsukuba, central Japan. The rate of ectomycorrhizal root tips was not different between heavily and lightly damaged pine stands. For ectomycorrhizae, Cenococcum geophilum had high relative abundance in the heavily damaged pine stand. The number of ectomycorrhizal fungal fruit bodies in the heavily damaged pine stand was much lower than that in the lightly damaged pine stands.     

Effect of ectomycorrhizal composition on survival and growth of <Emphasis Type="Italic">Pinus thunbergii</Emphasis> seedlings varying in resistance to the pine wilt nematode

Key message

Ectomycorrhizal composition and associated fungi affect the intra-specific ability of resistant black pines for physiological adaptation.

Abstract

Since Japanese black pine (Pinus thunbergii Parl.) forests have been widely devastated by pine wilt disease, several kinds of resistant black pines have been developed. Although all of the resistant black pines are the same species, these resistant trees show different physiological characteristics. We investigated the survival rates and growth rates, as well as ectomycorrhizal composition and associated fungi, on four kinds of Japanese black pine seedlings (three pine wilt-resistant and one non-resistant), and elucidated the factors affecting the various physiological characteristics. We found that the abundance of ectomycorrhizal types differed even though seedlings were grown sympatrically in the same areas for about 2 years. The seedlings that had plentiful white ectomycorrhizae showed the highest survival and growth rates regardless of the variety of black pine. Sequence similarities of the white ectomycorrhizae in the rDNA ITS region were best matched with members of Astraeus sp., Atheliaceae, Boletaceae and Thelephoraceae. Our findings indicate that intra-specific physiological adaptation might be affected by ectomycorrhizal composition or by the specific ectomycorrhizal species.

     

七株外生菌根真菌与三种松苗菌根的形成能力

松树外生菌根菌资源丰富,但实际应用的种类不多。为筛选出与松苗形成菌根能力较强的菌种,采用播种接菌和芽苗截根移栽接菌两种方法,对7株外生菌根真菌与马尾松、湿地松和黑松3种松苗的菌根合成进行了研究。结果表明：形成的菌根以二叉分枝状为主,棒状菌根相对较少,多叉状菌根以马尾松较多。PC2形成的菌根表面菌丝厚且紧密,504、EG、Pt,形成的菌根表面菌丝紧密程度中等,而505、ZJ和HX形成的菌根其表面菌丝则比较稀疏;Pt1、Pt2、EG形成的菌根外延菌丝较长,而505、HX形成的菌根外延菌丝极短。截根接菌时的感染率和感染指数要高于播种接菌。504形成菌根的能力最强,在3种松苗上的菌根感染率都达100％,感染指数最高可达90,最低也达70;Pt2和EG与马尾松和黑松形成菌根的能力较强;而505和HX仅与马尾松形成菌根的能力较强;Pt1形成菌根的能力较差,在3种松苗上菌根感染率和感染指数都较低。在3种松苗中,马尾松的菌根化状况最好,其次为黑松,湿地松的菌根化状况较差。     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

An in vitro method for establishing mycorrhizae on coniferous tree seedlings

Summary A method for in vitro synthesis of mycorrhizae on coniferous tree seedlings is described. Tree seedlings (Larix decidua Mill., Picea abies (L.) Karst, and Pinus sylvestris L.) and fungi Amanita muscaria (L. ex Fr.) Hooker, Piloderma croceum Erikss. et Hjorst., Pisolithus tinctorius (Pers.) Coker et Couch, and Suillus grevillei (Klotzsch) Singer were maintained under sterile conditions in petri dishes. Typical ectomycorrhizae were established within 2–3 weeks after inoculation and within 2 months after germination of seedlings. Eventually a high percentage of mycorrhizal root tips was obtained.     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant Gilbertiodendron dewevrei rain forest in Cameroon

Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests and are often invoked as a potential contributor to monodominance. We identified ectomycorrhizal fungi in a monodominant Gilbertiodendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermediate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic (¹³C and ¹⁵N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps) than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations suggest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the first report of the Tuber‐Helvella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.     

The extraction and quantification of ergosterol from ectomycorrhizal fungi and roots

Recently, ergosterol analysis has been used to quantify viable fungal biomass in resynthesized ectomycorrhizae. An objective of our study was to quantify ergosterol in a range of ectomycorrhizal isolates under differing growth conditions. In addition, we tested the applicability of the method on field-collected roots of ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal plants. Quantification of sitosterol as a biomass indicator of plant roots was also undertaken. Ergosterol was not detected in roots of uninoculated Betula populifolia seedlings, and sitosterol was not detected in an ectomycorrhizal fungal isolate but was present in birch roots. Ergosterol was produced in all isolates examined, which represented the major orders of ectomycorrhizal fungi. The range of values obtained, from 3 to nearly 18 g ergosterol mg^-1 dry mass, agrees well with reported values for other mycorrhizal and decomposer fungi. Hyphal ergosterol was the same during growth on phytic acid and KH₂PO₄. Reduction of growth temperature from 25° C to 15° C had little effect on ergosterol content of cultures harvested at similar growth stages. Ergosterol and sitosterol were detected in field-collected ectomycorrhizae of B. populifolia and Pinus sylvestris and VA mycorrhizae of Acer rubrum and Plantago major. Both ergosterol content and ergosterol to sitosterol ratios were significantly lower in VA mycorrhizae than ectomycorrhizae. Calculations of viable fungal biomass associated with field-collected roots were in agreement with those reported by others using the method on resynthesized ectomycorrhizae. Estimates of total mass could be obtained for field-collected B. populifolia roots by a simultaneously using ergosterol to estimate fungal biomass and sitosterol to estimate root mass. Some potential applications and limitations of sterol quantification in studies of mycorrhizal physiology and ecology are discussed.     

Comparative effects of four mycorrhizal fungi on loblolly pine seedlings growing in a greenhouse in a Piedmont soil

Summary A greenhouse study was conducted to evaluate the effect of ectomycorrhizae on loblolly pine (Pinus taeda L.) growing in a Piedmont soil. Pine seedlings were inoculated with one of four species of fungi (Scleroderma aurantium, Pisolithus tinctorius, Thelophora terrestris, andRhizopogon roseolus). The seedlings were grown in pots containing a Cecil sandy clay loam amended to create a gradient of extractable P ranging from 5.9 to 52.5 g/g. After ten months, all colonized seedlings were significantly larger than control seedlings. However, of the four fungi,Scleroderma aurantium mediated a far superior shoot growth response to increasing levels of soil P; the seedlings were significantly larger than those colonized by any other fungus and also had the largest root systems and greatest degree of mycorrhizal colonization.     

Salinity effects on germination,growth, and seed production of the halophyte Cakile maritima

The growth ofEucalyptus regnans seedlings in forest soil is enhanced when it has been air-dried. In undried forest soil seedlings grow poorly and develop purple coloration in the foliage, indicating P deficiency. This paper reports the results of pot experiments designed to investigate the relationship between growth and P acquisition, ectomycorrhizal infection and age of seedlings grown in air-dried and undried soil. The effect on seedling growth of their inoculation with air-dried or undried soil or with ectomycorrhizal roots from plants growing in air-dried or undried soil was also investigated. Ectomycorrhizal root tips were detected in 3-week-oldE. regnans seedlings in both air-dried and undried soil, and from then on the frequency of ectomycorrhizal root tips increased rapidly. In air-dried soil, seedlings were fully ectomycorrhizal at 9 weeks, and the occurrence of maximum ectomycorrhizal infection coincided with enhanced P acquisition and the initiation of rapid seedling growth. In undried forest soil, seedling growth remained poor, even though the seedlings had well-developed ectomycorrhizae and the incidence of ectomycorrhizal root tips was the same as in air-dried soil. The dominant ectomycorrhizae in airdried soil were associated with an ascomycete fungus, whereas in undried, undisturbed soil they were commonly associated with basidiomycete fungi. Inoculation of sterile soil/sand mix with washed ectomycorrhizal roots from air-dried soil increased the P acquisition and growth of the seedlings significantly compared with controls, whereas ectomycorrhizal inocula from undried soil had no effect on seedling growth, although both inocula resulted in a similar incidence of ectomycorrhizal root tips. Similarly, addition of a small amount of air-dried soil into sterile soil/sand mix resulted in a significantly greater increase in the P content and dry weight of the seedlings, compared with the control, than addition of undried soil. In both treatments, the incidence of ectomycorrhial root tips was similar. As (i) the differentiation in seedling growth between air-dried and undried soil occurred after seedlings became ectomycorrhizal, (ii) the dominant ectomycorrhizae in air-dried soil were different from those in undried soil, and (iii) inocula from air-dried soil, but not from undried soil, stimulated seedling growth in sterile soil/sand mix, it is concluded that development of particular ectomycorrhizae may be involved in seedling growth stimulation and enhanced P acquisition associated with air drying of forest soil.     

Effects of defoliation and symbiosis on polyamine levels in pine and birch

 We report the effect of ectomycorrhizal fungi (Suillus variegatus, Paxillus involutus) and defoliation on polyamine concentrations in pine (Pinus silvestris) and birch (Betula pendula) foliage and roots. Symbiotic root tips showed consistently higher concentrations of putrescine than non-symbiotic roots. Partial defoliation had no effect on the polyamine levels in mycorrhizal pine or birch roots. The foliage of mycorrhizal pine seedlings had lower putrescine concentrations and higher spermidine than foliage of non-mycorrhizal plants, and defoliation reversed this pattern. The response to partial defoliation differed in birch foliage: mycorrhizal status had no effect and all new growth after defoliation had higher spermidine levels than in non-defoliated birch. The potential role of polyamines in mycorrhizal symbiosis is discussed. Accepted: 26 February 1997     

The influence of mycorrhizal inoculation and paclobutrazol on water and nutritional status of Arbutus unedo L.

The purpose of this study was to analyze morphological and physiological aspects of Arbutus unedo L. plants treated with paclobutrazol (PAC), compounds characterized by their double activity as plant growth regulators and fungicides, and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch, which forms a special type of mycorrhizal colonization called arbutoid mycorrhiza. Native A. unedo L. seedlings were grown in a greenhouse and subjected to four treatments for 4 months: 0 or 60 mg of PAC and inoculated or not with P. tinctorius (Pers.). The arbutoid mycorrhizal inoculation increased in plants treated with PAC. Paclobutrazol reduced shoot and root biomass, plant height, internode length, stem diameter, leaf area, total root length and number of tips. P. tinctorius increased plant height and had a beneficial effect on the root system (increasing root diameter and the number of tips). PAC treatment led to an increase in ion levels in the leaf tissue, while mycorrhizal inoculation induced lower K and higher P contents in the roots. Leaf water potentials (at predawn and at midday) increased with the combined treatment. The absence of water deficit conditions meant there was no osmotic adjustment. Higher photosynthesis (P_n) values were associated with higher stomatal conductance (g_s) values in the mycorrhizal plants, which influenced water uptake from the roots. However, g_s decreased in the PAC-treated plants, reducing photosynthesis and, as a consequence, growth. The higher hydraulic conductivity (L_p) in the plants treated with PAC may have induced a better water energy status and good water transport. The combined treatment produced beneficial effects in the plants, improving their water and nutritional status.     

接种外生菌根对辽东栎幼苗生长的影响

 辽东栎（Quercus liaotungensis）是中国特有的栎林树种,也是中国暖温带落叶阔叶林的主要优势树种之一。铆钉菇（Gomphidius viscidus）和臭红菇（Russula foetens）是在自然环境中与其共生形成外生菌根的真菌。在温室花盆中播种辽东栎种子获得辽东栎幼苗,并对幼苗接种铆钉菇和臭红菇合成外生菌根,比较了有菌根和无菌根辽东栎幼苗生长、光合蒸腾特性、氮磷含量的差异。外生菌根对辽东栎幼苗的生长有明显的促进作用,有菌根幼苗的生物量、株高、净光合速率和水分利用效率高于无菌根幼苗,蒸腾速率则相反。有菌根幼苗的氮磷含量分别为无菌根幼苗的1.7倍和2.2倍,外生菌根的合成还改变了氮磷在幼苗器官间的分配比例,与无菌根幼苗相比,有菌根幼苗茎中的氮磷减少,而叶片中的磷显著增加。同时接种铆钉菇和臭红菇的生长促进效果优于单独接种。     

接种外生菌根对辽东栎幼苗生长的影响

辽东栎 (Quercusliaotungensis)是中国特有的栎林树种 ,也是中国暖温带落叶阔叶林的主要优势树种之一。铆钉菇 (Gomphidiusviscidus)和臭红菇 (Russulafoetens)是在自然环境中与其共生形成外生菌根的真菌。在温室花盆中播种辽东栎种子获得辽东栎幼苗 ,并对幼苗接种铆钉菇和臭红菇合成外生菌根 ,比较了有菌根和无菌根辽东栎幼苗生长、光合蒸腾特性、氮磷含量的差异。外生菌根对辽东栎幼苗的生长有明显的促进作用 ,有菌根幼苗的生物量、株高、净光合速率和水分利用效率高于无菌根幼苗 ,蒸腾速率则相反。有菌根幼苗的氮磷含量分别为无菌根幼苗的 1.7倍和 2 .2倍 ,外生菌根的合成还改变了氮磷在幼苗器官间的分配比例 ,与无菌根幼苗相比 ,有菌根幼苗茎中的氮磷减少 ,而叶片中的磷显著增加。同时接种铆钉菇和臭红菇的生长促进效果优于单独接种。