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Effects of organic fertilizer with or without a microbial inoculant on the growth and quality of lettuce in an NFT hydroponic system

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  • Organic food continues to increase in popularity worldwide. Similarly, hydroponic production of leafy greens is expanding globally and is an important component of the world's food supply. The purpose of this study was to evaluate the growth and quality of lettuce using six nutrient film technique (NFT) hydroponic systems. There were three treatments: organic fertilizer with or without a microbial inoculant and a conventional inorganic fertilizer as a control. The experiment was repeated over time. Results showed that the plants grown with organic fertilizer with additional microbial inoculant achieved similar shoot fresh and dry weight to those of the control, and dry weight was 17% higher than the organic fertilizer without inoculant. Nitrogen content in the shoot tissue of plants treated with organic fertilizer with inoculant was 10% and 24% greater than the control and the organic fertilizer without inoculant, respectively. However, when the organic fertilizer with inoculant was reused in a second experiment, shoot fresh and dry weight of plants in organic fertilizer with inoculant was lower than those in the control but were still higher compared to the organic fertilizer without inoculant. Additionally, electrical conductivity (EC) and pH of the organic fertilizer solutions fluctuated widely. Interestingly, relative chlorophyll content measured as SPAD and anthocyanin content in the leaf tissue increased in plants treated with organic fertilizer, regardless of inoculant, by 19% and 9%, respectively.
  • Bletilla Rchb. f. is one of the most economically valuable groups of orchids in the world. Due to its ornamental significance, the genus Bletilla occupies an important place in the worldwide horticultural market. Furthermore, in China, Japan, South Korea, and other Asian countries, it is highly valued for its medicinal use[1].

    There are eight species in the genus Bletilla, including Bletilla chartacea (King & Pantl.) Tang & F.T. Wang, Bletilla cotoensis Schltr., Bletilla foliosa (King & Pantl.) Tang & F.T. Wang, Bletilla formosana Schltr., Bletilla guizhouensis J. Huang & G.Z. Chen, Bletilla morrisonensis Schltr., Bletilla ochracea Schltr., and Bletilla striata Rchb.f.[2,3]. The distribution area spans from northern Myanmar in Asia to Japan via China[4]. Five species are native to China, namely, B. foliosa, B. formosana, B. guizhouensis, B. ochracea, and B. striata. In China, people have assigned various names to Bletilla based on its morphology and efficacy, such as baiji (白及/白芨), baigen (白根), baige (白给), baijier (白鸡儿), baijiwa (白鸡娃), diluosi (地螺丝), gangen (甘根), junkouyao (皲口药), lianjicao (连及草), and yangjiaoqi (羊角七)[5]. These diverse appellations highlight the importance of this genus in Chinese folk biological culture.

    The medicinal material known as 'baiji' in traditional Chinese medicine (TCM) is usually the dried tuber of B. striata, which is also the authentic product included in the Chinese Pharmacopoeia[6]. According to the Chinese Pharmacopoeia (2020), TCM baiji is sliced, dried, and crushed into a powder that can be used topically or internally, with a recommended dosage of 3–6 g at a time, offering astringent, hemostatic, detumescence, and myogenic effects. It is often used for conditions such as hemoptysis, hematemesis, traumatic bleeding, sores, and skin chaps[7]. Although only B. striata is the authentic product of TCM baiji, the other four Bletilla species native to China are also used as substitutes, and this practice is widespread[8].

    Modern research indicates that Bletilla contains a variety of chemical components, including benzol, dihydrophenanthrene, phenanthrene, and quinone derivatives. These components confer pharmacological effects on Bletilla, such as hemostasis, anti-tumor activity, and promotion of cell growth[9]. Due to its outstanding medicinal value, Bletilla can be found in nearly every corner of the traditional medicine market (Fig. 1). However, habitat destruction and uncontrolled mining have led to a significant reduction in the native populations of Bletilla, making its protection an urgent priority. Therefore, this paper provides a comprehensive review of relevant research up to August 2023, covering botanical characteristics, resource distribution, ethnobotanical uses, chemical components, pharmacological effects, clinical applications, and safety evaluations of Bletilla. The aim is to raise awareness and promote the protection and sustainable use of this genus.

    Figure 1.  Varieties of Bletilla at the traditional March Medicinal Market in Dali, Yunnan, China.

    The morphology of different Bletilla species is highly similar. The primary taxonomic feature distinguishing each species is the characteristics of the flower, particularly the lip of the flower, including its size, shape, and the number and shape of longitudinal ridges on the lip plate (Table 1, Fig. 2)[1014].

    Table 1.  The morphological differences among five species of Bletilla plants native to China.
    Morphological featureBletilla striataBletilla formosanaBletilla ochraceaBletilla foliosaBletilla guizhouensis
    Plant height (cm)18−6015−8025−5515−2045−60
    Rhizome shapeCompressedCompressedSomewhat compressedSubgloboseCompressed
    Rhizome diameter (cm)1−31−2About 21−1.53−4
    Stem characteristicsStoutEnclosed by sheathsStoutStout, shortThin
    Leaf shapeNarrowly oblongLinear-lanceolateOblong-lanceolateElliptic-lanceolateNarrowly lanceolate
    Leaf size (cm)8−29 × 1.5−46−40 × 0.5−4.58−35 × 1.5−2.85−12 × 0.8−325−45 × 1.2−4.5
    Flower colorPurplish red or pinkPale purple or pinkYellowPale purpleDeep purple
    Flower sizeLargeMediumMediumSmall to mediumLarge
    Inflorescence structureBranched or simpleBranched or simpleSimpleSimpleBranched
    Pedicel and ovary length (mm)10−248−12About 187−913−17
    Sepal shapeNarrowly oblongLanceolateLanceolateLinear-lanceolateOblong-elliptic
    Petal shapeSlightly larger than sepalsSlightly narrower than sepalsObliqueLanceolateOblong-elliptic
    Lip shapeObovate-ellipticBroadly ellipticNarrowly rhombic-obovateNarrowly oblongNarrowly oblong
    Lip colorWhite with purplish veinsWhitish to pale yellow with small dark purple spotsWhitish to pale yellow with small dark purple spotsWhite with purplish spots and purple edgeWhite with deep purple edge
    Number of lip Lamellae5 lamellae5 undulate lamellae5 longitudinal lamellae3 fimbriate lamellae7 longitudinal lamellae
    Column characteristicsSubterete, dilated towards apexSubterete, dilated towards apexSlender, dilated towards apexCylindric, dilated towards apexSuberect, with narrow wings
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    Figure 2.  (a)−(d) Bletilla striata (Thunb. ex Murray) Rchb. f. (e)−(h) Bletilla formosana (Hayata) Schltr. (i)−(l) Bletilla ochracea Schltr. (m), (n) Bletilla sinensis (Rolf) Schltr. (o), (p) Bletilla guizhouensis Jie Huang & G.Z. Chen (Photographed by Wang Meina, Zhu Xinxin, and He Songhua).

    The flowers of B. striata are large and purplish-red or pink, with narrowly oblong sepals and petals measuring 25−30 mm in length and 6−8 mm in width. They have acute apices, nearly as long as the sepals and petals. The lip is obovate or elliptic, predominantly white with purplish-red coloration and purple veins, measuring 23−28 mm in length, slightly shorter than the sepals and petals. The lip disc exhibits five longitudinal folds extending from the base to near the apex of the middle lobe, with waviness occurring only above the middle lobe[11]. In China, B. striata is found in regions such as Anhui, Fujian, Guangdong, Guangxi, Gansu, Guizhou, Hubei, Hunan, Jiangsu, Jiangxi, Shaanxi, Sichuan, and Zhejiang. It also occurs in the Korean Peninsula and Japan, thriving in evergreen broad-leaved forests, coniferous forests, roadside grassy areas, or rock crevices, at altitudes ranging from 100−3,200 m[12].

    B. ochracea's flowers are medium to large, featuring yellow or yellow-green exteriors on the sepals and petals, while the insides are yellow-white, occasionally nearly white. The sepals and petals are nearly equal in length, oblong, measuring 18−23 mm long and 5−7 mm wide, with obtuse or slightly pointed apices, often adorned with fine purple spots on the reverse side. The lip is elliptic, typically white or light yellow, measuring 15−20 mm in length and 8−12 mm in width, with three lobes above the middle. The lip disc is characterized by five longitudinally ridged pleats, with undulations primarily occurring above the middle lobe[13]. B. ochracea is native to southeastern Gansu, southern Shaanxi, Henan, Hubei, Hunan, Guangxi, Guizhou, Sichuan, and Yunnan, thriving in evergreen broad-leaved forests, coniferous forests, or beneath shrubs, in grassy areas or alongside ditches at altitudes ranging from 300−2,350 m[14].

    B. formosana's flowers come in shades of lavender or pink, occasionally white, and are relatively small. The sepals and petals are narrowly oblong, measuring 15−21 mm in length and 4−6.5 mm in width, and are nearly equal in size. The sepals have subacute apices, while the petal apices are slightly obtuse. The lip is elliptic, measuring 15−18 mm in length and 8−9 mm in width, with three lobes above the middle. The lip disc exhibits five longitudinal ridge-like pleats, which are wavy from the base to the top of the middle lobe[15]. B. formosana is indigenous to southern Shaanxi, southeastern Gansu, Jiangxi, Taiwan, Guangxi, Sichuan, Guizhou, central to northwest Yunnan, southeast Tibet (Chayu), and Japan. It is typically found in evergreen broad-leaved forests, coniferous forests, road verges, valley grasslands, grassy slopes, and rock crevices, at altitudes ranging from 600−3,100 m[16].

    The flowers of B. foliosa are small and lavender, with white sepals and petals featuring purple apices. The sepals are linear-lanceolate, measuring 11−13 mm in length and 3 mm in width, with subacute apices. The petals are lanceolate, also measuring 11−13 mm in length and 3 mm in width, with acute apices. The lip is white, oblong, adorned with fine spots, and features a purple apex. It measures 11−13 mm in length and 5−6 mm in width, tapering near the base and forming a scaphoid shape. The lip is anteriorly attenuated, unlobed, or abruptly narrowing with inconspicuous three lobes and exhibits fringe-like fine serrations along the edge. Three longitudinal ridge-like pleats are present on the upper lip disc[17]. B. foliosa typically grows on hillside forests, with its type specimen collected from Mengzi City, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, China[17].

    B. guizhouensis is a recently discovered species in Guizhou, China. In terms of shape, B. guizhouensis closely resembles B. striata, but it can be distinguished by its ovate-oblong buds, oblong dorsal sepals, obovate lips, and middle lobes of the lips, which are oval in shape. The disc of B. guizhouensis features seven distinct longitudinal lamellae, setting it apart from other known Bletilla species and establishing it as a distinct species[2]. Presently, B. guizhouensis has only been found in Guizhou, China, primarily thriving in evergreen broad-leaved forests at altitudes ranging from 900−1,200 m[3].

    Understanding the morphology, habitat, and distribution of Bletilla species is crucial for the conservation and propagation of these resources. To effectively implement plant conservation and breeding programs, a comprehensive understanding of the specific morphological characteristics, growth environments, and native habitats of these plants is essential, as without this knowledge, effective results cannot be achieved.

    The ethnobotanical uses of Bletilla worldwide primarily fall into two categories: ornamental and medicinal purposes. Bletilla orchids, renowned for their striking and distinct flowers, are commonly cultivated for ornamental purposes across many countries[18]. Valued for their aesthetic appeal, these orchids are frequently grown in gardens and utilized as potted plants. Among the various cultivars, B. striata stands out as the most favored choice for ornamental horticulture due to its ease of cultivation and adaptability to diverse climates[19,20].

    Contrastingly, in select Asian countries, Bletilla assumes a crucial role as a medicinal plant. For instance, influenced by TCM, the tuber of Bletilla also serves as a crude drug for hemostatic and anti-swelling purposes in Japan[21]. Likewise, traditional Korean medicine, deeply rooted in TCM principles, extensively documents the versatile use of Bletilla in addressing issues such as alimentary canal mucosal damage, ulcers, bleeding, bruises, and burns[22]. In Vietnam, Bletilla has been used as a medicinal herb for treating tumors and skin fissures, aligning with practices observed in the ethnic communities of southwest China[23].

    In China, Bletilla boasts a longstanding medicinal history, with numerous classical ancient Chinese medicine books containing detailed records of its medicinal applications[2432]. Even in contemporary society, many ethnic groups residing in mountainous areas in China continue to uphold the traditional medical practice of using Bletilla medicinally[31].

    In ancient Chinese medical literature, detailed records of Bletilla's morphology can be traced back to the late Han Dynasty, around 200 AD[24]. The Mingyi Bielu, a historical source, documented, 'Bletilla grows in the valley, with leaves resembling those of Veratrum nigrum L., and its root is white and interconnected. The ideal time for harvesting is September'. As awareness of the medicinal significance of Bletilla grew, successive dynastic-era Chinese medical texts consistently included descriptions of Bletilla's morphology (Table 2). In the Ming Dynasty, Li Shizhen compiled these earlier accounts of Bletilla's plant characteristics in his work, the Compendium of Materia Medica. He even provided an illustrative depiction of this plant genus (Fig. 3)[25].

    Table 2.  Morphological description of the plants belonging to Bletilla in the ancientChinese medicinal books.
    Dynasty (Year)TitleAuthorOriginal ChineseEnglish translation
    Late Han
    (184−220 AD)
    Mingyi Bielu/白给生山谷, 叶如藜芦,
    根白相连, 九月采
    Bletilla grows in the valley, with leaves like Veratrum nigrum L., root is white and connected. September is the time for harvesting.
    Wei-Jin period
    (220−420 AD)
    WuPu BencaoWu Pu白根, 茎叶如生姜, 藜芦,
    十月花, 直上, 紫赤色,
    根白连, 二月, 八月, 九月采
    Bletilla, stems and leaves like Zingiber officinale Roscoe and V. nigrum. It blooms in October and is purple and red, the inflorescence is vertical and upward. The roots are white and connected. It can be dug in February, August, and September.
    the Northern and Southern
    (420−589 AD)
    Bencao JizhuTao Hongjing近道处处有之, 叶似杜若,
    根形似菱米, 节间有毛
    It is everywhere near the road. The leaves are like Pollia japonica Thunb. The roots are like the fruit of Trapa natans L., and internode are many fibrous roots.
    Tang
    (618−907 AD)
    Su Jing, Zhangsun Wuji, etcTang materia medica生山谷, 如藜芦, 根白连, 九月采Born in the valley, with leaves like V. nigrum, root is white and connected. September is the time for harvesting.
    Song
    (960−1279 AD)
    Su SongCommentaries on the Illustrations白芨, 生石山上。春生苗,
    长一尺许, 似栟榈及藜芦,
    茎端生一台, 叶两指大, 青色,
    夏开花紫, 七月结实, 至熟黄黑色。
    至冬叶凋。根似菱米, 有三角白色, 角端生芽。二月, 七月采根
    Bletilla grow on the stone hill. It sprouts in spring and grows about a foot long. The seedlings are like Trachycarpus fortunei (Hook.) H. Wendl. and V. nigrum. The leaves are two finger-size. In summer, it blooms purple flowers and bears fruit in July. The ripe fruit is yellow-black. The leaves wither in winter. The root is like the fruit of T. natans, with three corners, white, and sprouting at the corners. The roots are dug in February and July.
    Ming
    (1368−1644 AD)
    Li ShizhenCompendium of Materia Medica一棵只抽一茎, 开花长寸许,
    红紫色, 中心如舌, 其根如菱米,
    有脐, 如凫茈之脐,
    又如扁扁螺旋纹, 性难干
    Only one stem per herb. The flower is more than one inch long, red and purple, and the center resembles a tongue. Its root is similar to the fruit of T. natans, possessing an umbilicus akin to that of Eleocharis dulcis (N. L. Burman) Trinius ex Henschel. It has spiral veins and is challenging to dry.
    −, Anonymous.
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    Figure 3.  Bletilla in Compendium of Materia Medica.

    Generally, ancient Chinese medical texts did not make clear distinctions between different Bletilla species. They collectively referred to plants with similar morphological traits as 'baige', 'baigen', 'baiji', 'gangen', 'lianjicao', or 'ruolan'. However, through textual analysis, it has been established that the descriptions of Bletilla in ancient texts before the Ming Dynasty largely align with Bletilla striata in terms of plant height, pseudo-bulb shape, leaf morphology, flower and fruit colors, and other characteristics. While the Bletilla portrayed in attached images may not precisely match B. striata in terms of morphology, considering the textual descriptions, it generally corresponds with B. striata. In writings from the Ming Dynasty and later periods, more specific descriptions of Bletilla emerged, encompassing details about its vascular arrangement, inflorescence, and flower structure, which consistently align with B. striata. Consequently, researchers have corroborated that the original plant of Bletilla described in ancient texts is Bletilla striata[24,33].

    According to the ancient Chinese medicinal books, Bletilla was used to treat a wide variety of conditions, including coughing, bruising, and bleeding, but their most mentioned use in ancient Chinese texts is for skin whitening and freckle removal[25]. Since ancient times, Bletilla species have been used consistently for skin care and whitening, and there are many well-known skincare products related to Bletilla. These Chinese formulae with Bletilla are similar to modern facial masks, face creams, facial cleanser, hand creams and other skin care products[26].

    For example, a prescription for 'facial fat (面脂)' in Medical Secrets from the Royal Library (752 AD) is made by boiling Bletilla with other traditional ingredients, and is applied to the face, resulting in skin whitening, freckle and wrinkle removal[27]. The 'Angelica dahurica cream (白芷膏)' in the General Medical Collection of Royal Benevolence (1111−1125 AD) is reputed to whiten facial skin through a seven-day treatment regiment, and contains Bletilla as the main botanical ingredient along with Angelica dahurica[28]. Jingyue Quanshu (1563-1640 AD) also contains a prescription called 'Yurong powder (玉容散)' for facial skin care. 'Yurong powder' is made of a fine powder of Bletilla, Nardostachys jatamansi (D. Don) DC., Anthoxanthum nitens (Weber) Y. Schouten & Veldkamp and other herbs[29]. Washing the face with Yurong powder in the morning and evening every day is said to make a person's face white without blemishes (Fig. 4)[29].

    Figure 4.  Yurong powder made of Bletilla and other traditional Chinese medicines in Jingyue Quanshu.

    In addition, in ancient Chinese medicine texts, Bletilla is also a well-known medicine for treating hematemesis, hemoptysis and bruises[23]. According to Shennong's Classic of Materia Medica (25−220), grinding the white fungus into fine powder and taking it after mixing with rice soup can be effective for treating lung damage and hematemesis[30]. Among the Prescriptions for Universal Relief (1406), 18 traditional Chinese medicines, such as Bletilla, are used to make 'snake with raw meat cream', which is said to be useful to treat carbuncles and incised wounds[31]. There is also a record of Bletilla powder treating lung heat and hematemesis in the Collected Statements on the Herbal Foundation (1624)[32].

    In ancient Chinese medicinal texts, most Bletilla are said to be useful for lung injury and hemoptysis, epistaxis, metal-inflicted wounds, carbuncles, burns, chapped hands and feet, whitening and especially for skin care. In the ancient medicinal texts, Bletilla is used alone or mixed with other traditional Chinese medicines. It is usually used in the form of a powder. The various medicinal effects of Bletilla described in these ancient texts suggest the great potential of this genus in clinical application, especially in the market of skin care products and cosmetics.

    As a skin care herb praised by ancient medical classics, 11 ethnic minorities in China, such as Bai, Dai, De'ang, Jingpo, Lisu, Miao, Mongolian, Mulao, Tu, Wa, and Yi still retain the traditional habit of using Bletilla for skin care in their daily life (Table 3). In addition to B. striata, B. formosana and B. ochracea are also used as substitutes. Although Chinese ethnic groups have different names for Bletilla spp., the skin care methods are basically the same. Dry Bletilla tubers are ground into a powder and applied to the skin[34], and this usage is also confirmed by the records in ancient medical texts[23, 24]. The various local names of Bletilla by different ethnic groups also indirectly suggests which ethnic groups play an important role in the traditional use. For example, Bai people called B. striata baijier (白鸡儿), goubaiyou (狗白尤), and yangjiaoqi (羊角七) (Table 3).

    Table 3.  The traditional medicinal knowledge of Bletilla in ethnic communities, China.
    Ethnic groupLatin nameLocal nameUsed partUse methodMedicinal effect
    AchangBletilla striata (Thunb. ex Murray) Rchb. F.BaijiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationTuberculosis, hemoptysis, bleeding from gastric ulcer, burns and scalds
    BaiBaijier, Goubaiyou, YangjiaoqiTuberTreatment of tuberculosis hemoptysis, bronchiectasis hemoptysis, gastric ulcer hemoptysis, hematochezia, skin cracking
    DaiYahejieTuberUsed for tuberculosis, tracheitis, traumatic injury, and detumescence
    De'angBageraoTuberTuberculosis, hemoptysis, bleeding from gastric ulcer, burns and scalds
    DongShaque, SanjueTuberTreat hematemesis and hemoptysis
    JingpoLahoiban, PusehzuotuberFor tuberculosis, bronchiectasis, hemoptysis, gastric ulcer, hematemesis, hematuria, hematochezia, traumatic bleeding, burns, impotence
    MengMoheeryichagan, NixingTuberFor tuberculosis hemoptysis, ulcer bleeding, traumatic bleeding, chapped hands, and feet
    MiaoBigou, Wujiu, SigouTuberUsed for hemoptysis of tuberculosis, bleeding of ulcer disease, traumatic bleeding, chapped hands, and feet
    MolaoDajiebaTuberTreat internal and external injuries caused by falls
    TibetanSanchabaijiTuberFresh chopped and soaked with honey; Powdered after sun-dried, then taken with honey and waterMainly used to treat cough, asthma, bronchitis, lung disease and a few gynecological diseases
    TuRuokeyeTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationTreatment of tuberculosis, hemoptysis, bloody stool, chapped skin
    WaBaijiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationFor tuberculosis, hemoptysis, gastrointestinal bleeding, scald and burn
    YaoBiegeidaiTuberTreat gastric ulcer, pulmonary tuberculosis, cough, hemoptysis, and hematemesis
    YiDaibaij, Tanimobbaili, Niesunuoqi, AtuluoboTuberTreatment of tuberculosis, hemoptysis, golden wound bleeding, burns, chapped hands and feet
    ZhuangManggounuTuberTreat stomachache and hemoptysis
    BaiBletilla formosana (Hayata) Schltr.Baijier, YangjiaoqiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationIt is used for emesis, hemoptysis due to tuberculosis, and hemoptysis due to gastric ulcer. External application for treatment of incised wound
    MiaoLianwuTuberThe effect is the same as that of B. striata
    LisuHaibiqiuTuberIt can treat tuberculosis, hemoptysis, epistaxis, golden sore bleeding, carbuncle and swelling poison, scald by soup fire, chapped hands and feet
    YiNiesunuoqi, Yeruomaoranruo, Atuluobo, Ribumama, Atuxixi, Abaheiji, Binyue, ZiyouTuberIt is used for tuberculosis, hemoptysis, traumatic injury, treatment of frostbite, burn, scald, bed-wetting of children and other diseases
    BaiBletilla ochracea Schltr.Baijier, YangjiaoqiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationFor hematemesis, epistaxis, hemoptysis due to tuberculosis, hemoptysis due to gastric ulcer; External application of golden sore and carbuncle
    MengMoheeryichagan, NixingtuberThe effect is the same as that of B. striata
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    The formation of traditional medical knowledge among Chinese people is often directly related to the specific living environment and cultural background[34]. For example, the Bai, Dai, De'ang, Jingpo, Lisu Yi, Wa and other ethnic minorities live in mountainous areas. The cold weather in winter and year-round outdoor manual work makes it difficult to maintain their skin[35, 36]. In the face of this situation, the ethnic people who are concerned about their physical appearance have long ago chosen local Bletilla species for skin care, and have handed down this tradition for many generations[34]. This important traditional skin care tradition is worthy of further in-depth study.

    The six main classes of Bletilla chemical components, phenanthrene derivatives, phenolic acids, bibenzyls, flavonoids, triterpenoids, and steroids, have been described previously. Almost three hundred compounds have been isolated from Bletilla, including 116 phenanthrene derivatives, 58 phenolic acids, 70 bibenzyls, 8 flavonoids, 24 triterpenoids and steroid and 13 other compounds (Figs 514). Chemical structures of the isolates of Bletilla species most are phenanthrene derivatives, which have been demonstrated to possess various pharmacological activities.

    The prominent opioids oxycodone, hydrocodone, naloxone, and naltrexone are all phenanthrene derivatives[37]. Currently, phenanthrene derivatives (Fig. 5, 1 to 66) were isolated from B. formosana, B. ochracea, and B. striata. In 2022, 17 phenanthrene derivatives (117) were isolated from the ethyl acetate (EtOAc) extracts of B. striata tubers[38]. Then, other phenanthrene derivatives were isolated from Bletilla, such as dihydrophenanthrenes (1841), phenanthrenes (4266), biphenanthrenes (Fig. 6, 6789), dihydro/phenanthrenes with uniquestructures (90112) and phenanthraquinones (Fig. 7, 113116). Thus far, this genus has been documented to include these compounds, which have been shown to exhibit pharmacological actions[3945].

    Figure 5.  Phenanthrene derivatives from Bletilla species (1−66)[3841,4345,47,49,58,7072,7479].
    Figure 6.  Phenanthrene derivatives from Bletilla species (67−105)[41,43,49,5961,70,76,7986].
    Figure 7.  Phenanthrene derivatives from Bletilla species (106−116)[43,49,70,75,84,85,87].

    Phenolic acids are carboxylic acids created from the skeletons of either benzoic or cinnamic acids[4648]. Fifty-eight phenolic acids (Figs 810, 117 to 174) were isolated from B. formosana, B. ochracea, and B. striata.

    Figure 8.  Phenolic acids from Bletilla species (117−134)[1,5,36,4752,54,67,88,90].
    Figure 9.  Phenolic acids from Bletilla species (135−169)[39,45,4854,56,61,68,69,73,76,82,83,8994].
    Figure 10.  Phenolic acids from Bletilla species (170−174)[20,95].

    For example, compounds 121, 126, 139, 141, 148, 149, 154, 155 and 157 were isolated from the rhizomes of B. formosana[1,49,5052]. The structures of these compounds were determined, mostly from their NMR spectroscopy data. Additionally, protocatechuic (136) and vanillin (137) also have been isolated from B. striata[53]. Moreover, some bioactive components such as 2-hydroxysuccinic acid (164) and palmitic acid (165) have been discovered and identified from B. striata[20,5456].

    The bibenzyls were small-molecular substances with a wide range of sources, which were steroidal ethane derivatives and resembling the structural moiety of bioactive iso-quinoline alkaloids[57].

    For example, depending on their structural characteristics, 70 bibenzyl compounds (Fig. 11, 175 to 244) can be grouped into three groups, simple bibenzyls (175186, 233238), complex bibenzyls (189225) and chiral bibenzyls (226-232, 239-244)[5860].

    Figure 11.  Bibenzyls from Bletilla species (175-244)[1,4042,47,49,50,5860,70,73,76,9699].

    Flavonoids are among the most common plant pigments. Eight bibenzyls (Fig. 12, 245 to 252) have been isolated from B. formosana, B. ochracea, and B. striata. Apigenin (245) and 8-C-p-hydoxybenzylkaempferol (249) were isolated from the whole plant of B. formosana[45]. Bletillanol A (250), bletillanol B (251) and tupichinol A (252) were isolated from B. striata[61]. The names and chemical structures of the flavonoids reported from Bletilla are shown below (Fig. 12).

    Figure 12.  Flavonoids from Bletilla species (245–252)[45,61].

    Twenty-four triterpenoids and steroids (Fig. 13, 253 to 276) have been reported from Bletilla (Fig. 13), such as, tetracyclic triterpenes (253259) and pentacyclic triterpenes (189225) and chiral bibenzyls (260)[6264]. Steroids (261276) isolated from the Bletilla and have shown some bioactivity. For example, bletilnoside A (272) was isolated from Bletilla species and displayed anti-tumor activity[65,66].

    Figure 13.  Triterpenoids and steroid compounds from Bletilla species (253-276)[56,6265].

    Thirteen other compounds (Fig. 14, 277 to 289) were isolated from B. formosana, B. ochracea, and B. striata. These compounds included amino acids, indoles and anthraquinones[67,68]. For example, syringaresinol (285) and pinoresinol (286) have been described in the methanol extract of the tubers of B. striata[61].

    Figure 14.  Others compounds from Bletilla species (277−289)[50,54,61,62,67,68,94,97].

    Based on the information about the chemical constituents of Bletilla species, it appears that there is a substantial body of research on these compounds. However, there are some areas that may warrant further investigation and research. At first, it would be valuable to investigate potential synergistic effects and interactions between the different classes of compounds within Bletilla species, as some of the compounds may work together. Besides, it is worth considering the improvement of compound yield. Optimizing extraction methods and finding the most efficient and environmentally friendly techniques are vital for both research purposes and potential commercial applications. It is also important to take into account the variability in chemical composition among different Bletilla species and even within the same species from different cultivars.

    The rich and varied chemical components make the plants of Bletilla have a wide variety of pharmacological activities (Table 4). Many studies have shown that the plants of this genus have anti-inflammatory, antineoplastic, antiviral, antioxidant, hemostatic, antibacterial, and other biological activities, which help to support the traditional medicinal practice of Bletilla in folk medicine.

    Table 4.  Summary of the pharmacological activities of Bletilla species.
    Pharmacological activityTested substance/partTested system/organ/cellTested dose/dosing methodResultsRefs.
    Anti-inflammatoryEthanol extract of Bletilla striataRAW264.7 cells RAW264.7 cells were pre-treated with ethanol extract of B. striata for 1 h and then stimulated with LPS (200 ng/mL) for 12 h, 0.05% DMSO was applied as the parallel solvent control. The culture supernatant was collected for IL-6 and TNF-α detection.Ethanol extract of B striata significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 µg/mL.[41]
    The ethyl acetate-soluble (EtOAc) extract of tubers of B. striataH2O2-induced PC12 cell injury modelPC12 cells were seeded in 96-well multiplates at a density of 1.5 × 105 cells/mL. After overnight incubation at 37 °C with 5% CO2, 10 μM test samples and H2O2 (final concentration of 450 μM) were added into the wells and incubated for another 12 h.It protected the cells with the cell viabilities of 57.86 ± 2.08%, 64.82 ± 2.84%, and
    64.11 ± 2.52%.
    [98]
    Ethanol extract of tubers of B. striataRAW264.7 cellsCells were treated with ethanol extracts (25 μM) dissolved in DMSO, in the presence of
    1 μg/mL lipopolysacchride
    (LPS) for 18 h
    The anti-inflammatory activity with IC50 of 2.86 ± 0.17 μM.[54]
    PE extract of the tubers of B. striataLPS-stimulated BV2 cellsCells treated with extract
    (0, 1, 10, 30, 100 μg/mL) and dihydropinosylvi (0, 1, 10, 30, 100 μM) in presence of LPS
    (1 μg/mL)
    The anti-inflammatory activity with IC50 values of 96.0 μM.[96]
    Ethanol extract of the roots of B. striataCox-1 and Cox-2Treated with the ethanol extracts at various concentrations
    (0, 1, 10, 100 μM)
    The compounds with sugar moieties displayed selective inhibition of Cox-2 (N90%).[38]
    B. striata polysaccharide (BSPb)Human mesangial cells (HMCs)HMCs were pre-treated with BSPb (5, 10, 20 μg/mL)BSPb efficiently mediated expression of NOX4 and TLR2, to attenuate generation of ROS and inflammatory cytokines.[12]
    Compounds extracted from the rhizomes of Bletilla ochraceaRAW264.7 cellsAfter 24 h preincubation,
    cells were treated with serial dilutions in the presence of
    1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in the supernatant was assessed by adding 100 μL of Griess regents.
    It showed the inhibitory effects with IC50 values in the range of 15.29–24.02 μM.[76]
    Compounds extracted from the rhizomes of B. ochraceaMurine monocytic RAW264.7 cellsAfter 24 h preinubation, RAW 264.7 cells were treated with compounds (25 μM) dissolved in DMSO, in the prenence of
    1 μg/mL LPS for 18 h. NO production in each well was assessed by adding 100 μL of Giress regent
    It showed the inhibitory effects with IC50 2.86 ± 0.17 μM.[86]
    Compounds extracted from the rhizomes of Bletilla formosanaElastase Release AssaysNeutrophils (6 × 105 cells/mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM) at 37 °C for 2 min and then incubated with a test compound or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min.Most of the isolated compounds were evaluated for their anti-inflammatory activities. The results showed that IC50 values for the inhibition of superoxide anion generation and elastase release ranged from 0.2 to 6.5 μM and 0.3 to 5.7 μM, respectively.[49]
    Anti-tumorTwo compounds from Bletilla striataA549 cellsCompounds were tested for their ability to induce ROS generation in A549 cells at concentrations of 20 two compounds for 24 h, the cells were harvested to evaluate the ROS production.The two compounds exhibited antiproliferative effects using the MTT test; these effects may be due to cell cycle arrest and inducing ROS generation.[87]
    Stilbenoids from B. striataBCRP-transduced K562 (K562/BCRP) cellsIt showed antimitotic activity and inhibited the polymerization of tubulin at IC50 10 μM.[78]
    Compounds extracted from the rhizomes of B. ochraceaThe human tumor cell lines HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer)100 μL of adherent cells were seeded into each well of 96-well cell culture plates. After 12 h of incubation at 37 °C, the test compound was added. After incubated for 48 h, cells were subjected to the MTS assay.All isolated metabolites except 7 were evaluated for cytotoxic activity against five human cancer cell lines (HL-60, SMMC7721, A-549, MCF-7 and SW480).[76]
    AntiviralThe tuber of B. striataMadin-Darby canine kidney model and embryonated eggs modelAs simultaneous treatment with 50% inhibition concentration (IC50) ranging from 14.6 ± 2.4 to 43.3 ± 5.3 μM.Phenanthrenes from B. striata had strong anti-influenza viral activity in both embryonated eggs and MDCK models.[107]
    The 95% ethanol
    Extract of B. striata
    BALB/C miceIt has significant anti-influenza
    virus effect in mice, which may be related to the increase of IL-2, INFα, INF-β and thus enhance the immune function of mice.
    [12]
    AntioxidantCompounds extracted from the rhizomes of B. formosanaDPPH radical-scavenging assaySolutions containing 160 μL of various concentrations of sample extract, 160 μL of various concentrations of BHA, 160 μL of various concentrations of ascorbic acid, and the control (160 μL of 75% methanol) were mixed separately with 40 μL of 0.8 mM DPPH dissolved in 75% methanol. Each mixture was shaken vigorously and left to stand for 30 min at room temperature in the dark.Tthe seedlings grown by tissue culture of B. formosan collected in Yilan County had the best antioxidant capacity. In addition, B. formosana collected in Taitung County had the best scavenging capacity in the tubers, leaves and roots.[93]
    Fibrous roots of B. striataDPPH model and superoxide anion systemThe ABTS+ solution was prepared by reacting 7 Mm ABTS with 2.45 mM potassium persulfate (final concentrations both dissolved in phosphate buffer, 0.2 M, pH 7.4) at room temperature for 12–16 h in the dark.It removed free radicals and inhibit tyrosinase activity.[33]
    B. striata extracts (BM60)The murine macrophage cells NR8383, male SD mice (180~200 g)NR8383 were pretreated with extracts (1, 10 and 100 g/mL) for 4 h and then 65 stimulated with 1 g/mL of LPS for 24 h. Acute lung injury was induced in mice by nonhexposure intratracheal instillation of LPS (3.0 mg/kg). Administration of the BM60 extract of 35, 70, and 140 mg/kg (L, M, H) was performed by oral gavages.The BM60 treatment reduced the production of NO in NR8383 macrophages. Treatments with BM60 at the doses of 35, 70, 140 mg/kg significantly reduced macrophages and
    neutrophils in the bronchoalveolar lavage fluid (BALF).
    [12]
    The crude
    polysaccharides obtained from B. striata
    DPPH free radical scavenging activityConcentration
    range of 2.5–5.0 mg/mL
    The IC50 of BSPs-H was 6.532 mg/mL.[35]
    HemostasisB. striata polysaccharide (BSP)Diabetes mellitus (DM) mouse models were induced by high fat-diet feeding combined with low-dose streptozocin injectionDM mouse models were induced by high fat-diet feeding combined with low-dose streptozocin injection. The BSP solutions were applied on the surface of each wound at a volume of 50 μl. RD mice were assigned as normal controls and received saline treatment (n = 6). All mice were treated with vehicle or BSP once daily from the day of wounding (d0) until 12 days later (d12).BSP administration accelerated diabetic wound healing, suppressed macrophage infiltration, promoted angiogenesis, suppressed NLRP3 inflammasome activation, decreased IL-1β secretion, and improved insulin sensitivity in wound tissues in DM mice.[112]
    B. striata Micron Particles (BSMPs)Tail amputation model and healthy male Sprague-Dawley (SD) rats
    (250 ± 20 g, 7 weeks of
    age)
    Rats were divided into six groups of five treated with cotton gauze and BSMPs (350–250, 250–180, 180–125, 125–75, and < 75 μm), respectively.Compared to other BSMPs of different size ranges, BSMPs of 350–250 μm are most efficient in hemostasis. As powder sizes decrease, the degree of aggregation between particles and hemostatic BSMP effects declines.[109]
    Rhizoma Bletillae polysaccharide (RBp)Adult male SD rats weighing 220 ± 20 gAfter incubation for 1 min at 37 °C, 300 μL of PRP was dealt with different concentrations of RBp (50, 100, 150, and 200 mg/L) under continuous stirring, and the vehicle was used as the blank control.RBp significantly enhanced the platelet aggregations at concentrations of 50−200 mg/L in a concentration-dependent manner.[113]
    AntibacterialBibenzyl derivatives from the tubers of Bletilla striataS. aureus ATCC 43300, Bacillus subtilis ATCC 6051, S. aureus ATCC 6538 and Escherichia coli ATCC 11775Using a microbroth dilution method, bacteria were seeded at
    1 × 106 cells per well (200 μL) in a
    96-well plate containing Mueller-Hinton broth with different concentrations (from 1 to 420 μg/mL, 300 μg/mL and so on;
    2-fold increments) of each test compound.
    It showed inhibitory activities with MIC of (3–28 μg/mL) against S. aureus ATCC6538[116]
    The crude extract of B. striataS. album, A. capillaris, C. cassiaThey were seeded at 1 × 106 cells per well (200 μL) in a 96-well plate containing Mueller−Hinton broth (meat extracts 0.2%, acid digest of casein 1.75%, starch 0.15%) with different concentrations (from 1 to 128 μg/mL; 2-fold increments) of each test compound.It showed S. album (0.10%), A. capillaris (0.10%), and C. cassia (0.10%) to have the strongest antibacterial properties.[118]
    The ethyl acetate-soluble (EtOAc) extract of tubers of B. striataS. aureus ATCC 43300, S. aureus ATCC 6538, and Bacillus subtilis ATCC 6051) and Escherichia coli ATCC 11775)Bacteria were seeded at 1 × 106 cells per well (200 μL) in a 96-well plate containing Mueller Hinton broth with different concentrations (from 1 to 420 μg/ml; 2-fold increments) of each test compound.The extract was effective against three Gram-positive bacteria with minimum inhibitory concentrations (MICs) of 52–105 μg/ml.[98]
    The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbsS. aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 43300, E. coli ATCC 35218, and P. aeruginosa ATCC 27853, Bacillus subtilis 168EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 μg/mL.EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively.[117]
    Anti-adhesiveBletilla striata extraction solutionPPA was induced by cecal wall abrasion, and Bletilla striata was injected to observe its efect on adhesion in ratsThe rats in the sham operation group was not treated; the other rats of the three experimental groups were intraperitoneally injected with 8 ml of phosphate-buffered saline (Control group), 15% Bletilla striata extraction solution (BS group), and 0.2% hyaluronic acid solution (HA group), respectively.Bletilla striata decreased the development of abdominal adhesion in abrasion-induced model of rats and reduced the expression of the important substance which increased in PPAs.[120]
    ImmunomodulatoryB. striata polysaccharide (BSPF2)Mouse spleen cellsTo observe the immune activity of BSPF2, mouse spleen cells were stimulated with BSPF2 at 10–100 g/mL for 72 h.Immunological assay results demonstrated that BSPF2 significantly induced the spleen cell proliferation in a dose-dependent manner.[121]
    Anti-pulmonary fibrosisB. striata polysaccharideClean grade male SD ratsSD rats were randomly divided into 5 groups, sham operation group (equal volume of normal saline), model group (equal volume of normal saline), tetrandrine positive control (24 mg/kg) group and white and Polysaccharide low
    (100 mg/kg) and high (400 mg/kg) dose groups.
    The Bletilla striata polysaccharide has remarkable regulation effect on anti-oxidation system and immune system, but cannot effectively prevent lung fibrosis.[127]
    Small molecule components of Bletilla striataClean grade male SD ratsSD rats were randomly divided into 5 groups, sham operation group (0.5 mL normal saline), model group (0.5 mL normal saline), and positive control group (tetrandrine 24 mg/kg) and low (20 mg/kg) and high (40 mg/kg) dosage groups of the small molecule pharmacological components of Bletilla, which were administered by gavage once a day for 2 consecutive months.The small molecule components of Bletilla striata can effectively prevent lung fibrosis though regulating the anti-oxidation system,immune system and cytokine level; SMCBS is one of the active components of Bletilla striata on silicosis therapy[124]
    —, not given.
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    Many phytochemicals have been well characterized to lessen swelling or inflammation[89]. A series of phenolic acid and polysaccharide compounds isolated from Bletilla demonstrated anti-inflammatory bioactivity against BV-2 microglial, RAW 264.7, and PC12 cells[96,100102]. For example, phochinenin K (106) exhibited growth inhibitory effects with an IC50 of 1.9 μM, and it is a possible candidate for development as neuroinflammation inhibitory agent[43]. Using the H2O2-induced PC12 cell injury model, (7S)-bletstrin E (242), (7R)-bletstrin F (243) and (7S)-bletstrin F (244) could clearly protect the cells with the cell viabilities of 57.86% ± 2.08%, 64.82% ± 2.84%, and 64.11% ± 2.52%, respectively[98]. With an IC50 of 2.86 ± 0.17 μM, 2,7-dihydroxy-4-methoxyphenanthrene (53) showed potential action against NO generation in RAW 264.7 macrophages[54]. The use of Bletilla in traditional skin care, it is said to function as an astringent, hemostatic and wound healing[33]. Modern medical pharmacology research has validated that this plant has antibacterial effects, which may may help to explain, in part, its traditional use in skin care[24].

    Though it's mentioned that some of these compounds from Bletilla have demonstrated anti-inflammatory action, more extensive studies are needed to fully understand their mechanisms of action, potential therapeutic applications, and safety profiles. Conducting in vivo studies and clinical trials can provide more concrete evidence of their effectiveness.

    There are important antineoplastic agents that have originated from plant natural products[103]. In recent years, several bibenzyl and flavonoid compounds have been discovered from Bletilla that have antineoplastic activity against A549 cells and other cells. For example, 7-hydroxy-2-methoxy-phenanthrene-3,4-dione (160) and 3′,7′,7-trihydroxy-2,2′,4′-trimethoxy-[1,8′-biphenanthrene]-3,4-dione (163) have shown strong antiproliferative effects and induced ROS production after 24 h in A549 cells[87]. The doxorubicin (Dox)/FA (folate)-BSP-SA (stearic acid) modified Bletilla striata polysaccharide micelles boosted the drug enrichment in tumors and improved the in vivo anticancer effects[104,105]. Micelles, nanoparticles, microspheres, and microneedles are examples of B. striata polysaccharide-based drug delivery systems that exhibit both drug delivery and anti-cancer functionality. These experiments confirmed that some of the compounds isolated from the Bletilla have potential activity for the treatment of cancer.

    However, most of the evidence presented in the previous studies is based on in vitro experiments or cell culture studies. It is highly necessary to use animal models to study the in vivo anti-tumor effects of Bletilla extracts or compounds. These studies can help evaluate the safety and effectiveness of treatments based on Bletilla. Additionally, through such methods, researchers can further investigate the mechanisms of Bletilla's anti-tumor activities, exploring how Bletilla compounds interact with cancer cells, immune responses, and signaling pathways involved in tumor growth and metastasis.

    Antiviral medications are essential for preventing the spread of illness, and are especially important nowadays with pandemics and drug-resistant viral strains[5, 6]. Therefore, it is vitally necessary to find novel, safe, and effective antiviral medications to treat or prevent viral infections[106]. B. striata plant contains compounds that have been recorded in ancient texts to cure cough, pneumonia, and skin rashes, and these may be related to potential antiviral constituents[23]. Some constituents of B. striata have antiviral activity, for example, phenanthrenes and diphenanthrenes from B. striata displayed potent anti-influenza viral in a Madin-Darby canine kidney model and embryonated eggs model, diphenanthrenes with parentally higher inhibitory activity than monophenanthrenes[107]. But more research is needed to further determine the antiviral activity of Bletilla, understand how Bletilla compounds interact with viral proteins or the host immune response, and conduct safety and toxicity studies, which are crucial for the development of related materials.

    Free radicals have the potential to exacerbate lipid peroxidation and harm cell membranes, which can lead to several prevalent human diseases, including cancer, cataracts, and coronary heart disease[108]. Research has shown that extracts from Bletilla possess strong antioxidant activity. However, this antioxidant activity can vary depending on the different growing environments of the plant. Additionally, the antioxidant capabilities of extracts from different parts of the Bletilla plant also vary[93]. Clinical studies have shown that traditional Chinese medicine formulas containing Bletilla can inhibit tyrosinase activity and possess antioxidant properties, thus resulting in skin-whitening effects[108]. Furthermore, some research reveals that the polysaccharides in the plant exhibit significant antioxidant activity, effectively scavenging free radicals and inhibiting tyrosinase activity[33]. This highlights the skin-whitening potential of the fibrous root of Bletilla striata, indicating promising prospects for the comprehensive utilization of the B. striata plant[33]. However, most studies on the pharmacological activities of Bletilla have focused solely on B. striata, neglecting other species within the genus. Different species may possess varying phytochemical compositions and antioxidant properties, which can lead to an incomplete understanding of the genus as a whole.

    Available hemostatic agents are expensive or raise safety concerns, and B. striata may serve as an inexpensive, natural, and promising alternative[109]. Polysaccharides of B. striata displayed hemostatic activity through inhibition of the NLRP3 inflammasome[110112]. The ADP receptor signaling pathways of P2Y1, P2Y12, and PKC receptors may be activated as part of the hemostasis[113]. Alkaloids from Bletilla have hemostatic activities through platelet deformation, aggregation, and secretion. In addition, polysaccharides of Bletilla striata have potential wound-healing medicinal value[110]. Currently, Bletilla plants have been used in various traditional systems, such as traditional Chinese medicine and Ayurveda, to control bleeding.

    Previous studies revealed that Bletilla displayed antibacterial effects[114]. For example, bletistrin F, showed inhibitory activities with MIC of (3–28 μg/mL) against S. aureus ATCC 6538[115,116]. Antimicrobial screening of Bletilla showed S. album (0.10%), A. capillaris (0.10%), and C. cassia (0.10%) to have the strongest antibacterial properties[117,118]. In addition, phenanthrenes are worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA[119]. However, further in vivo studies on the antibacterial activity of Bletilla are lacking, which is needed for clinical application. For example, the specific mechanism of antibacterial activity of Bletilla still needs to be elucidated. While research on the antibacterial activity of Bletilla plants is promising, it faces several shortcomings and challenges that need to be addressed for a more comprehensive understanding of their potential therapeutic applications. Further studies with standardized methodologies, mechanistic insights, clinical trials, and consideration of ecological and safety concerns are essential to advance this field.

    There are other pharmacological activities of Bletilla, like anti-fibrosis activity, anti-adhesive activity, and immunomodulatory activity. For example, B. striata has been studied as a new and cheaper antiadhesive substance which decreased the development of abdominal adhesion abrasion-induced model in rats[120]. However, the natural resources of Bletilla are also getting scarcer. To preserve the sustainable development of Bletilla species, proper farming practices are required, along with the protection and economical use of these resources. The immunomodulatory activity of the Bletilla species was assessed using the 3H-thymidine incorporation method test, and BSP-2 increased the pinocytic capacity and NO generation, which improved the immunomodulatory function[121,122].

    B. striata extract was shown to have anti-pulmonary fibrosis effect[123]. B. striata polysaccharide can successfully prevent lung fibrosis through established by invasive intratracheal instillation method and evaluated by lung indexes[123,124]. Moreover, Bletilla species need further investigations to evaluate their long-term in vivo and in vitro activity before proceeding to the development of pharmaceutical formulation.

    While there is currently a deep understanding of the pharmacological activity of plants in the Bletilla genus, there are still many gaps that need to be addressed. To overcome these shortcomings, future research on the pharmacological activity of Bletilla species should emphasize comprehensive, well-designed studies with a focus on species-specific effects, mechanistic insights, and rigorous clinical trials. Additionally, collaboration among researchers, standardization of methods, and transparent reporting of results can help advance our understanding of the therapeutic potential of Bletilla plants. Researchers should also consider safety aspects and explore potential herb-drug interactions to ensure the responsible use of Bletilla-based therapies.

    There are several common clinical applications of Bletilla striata in TCM. The gum of B. striata has unique viscosity characteristics and can be used as thickener, lubricant, emulsifier and moisturizer in the petroleum, food, medicine, and cosmetics industries[125130]. B. striata is used as a coupling agent, plasma substitute, preparation adjuvant, food preservative and daily chemical raw material[131133]. In clinical practice, B. striata glue has also been proven to control the infections and is beneficial to the healing of burns and wounds[133135].

    In ethnic communities in Southwest China, the locals chew fresh Bletilla tubers directly or take them orally after soaking in honey to treat cough, pneumonia and other diseases[33, 34]. This traditional use is common in local communities in Southwest China, and suggests at the safety of Bletilla. However, current research shows it is still necessary to control the dosage when using Bletilla[136].

    Zebrafish embryos and larvae respond to most drugs in a manner similar to humans[137]. Militarine, the main active ingredient of Bletilla, was tested in a zebrafish embryo development assay at concentrations of 0.025 g/L and 0.05 g/L, and with the increased concentration, the heart rate of zebrafish embryos is slowed. Mortality and malformation rates of zebrafish embryos gradually increased with time and militarine concentration[138]. Although Bletilla species are safe at therapeutic dose ranges, further research on their safety is required[136]. More in-depth studies should be carried out on Bletilla to extract effective ingredients and make better preparations for clinical use[139].

    According to the traditional medicinal knowledge in ancient Chinese texts, Bletilla has been an important ingredient for skin care since ancient times. Many ethnic minority groups in China still retain the practice of using Bletilla for skin care, and the plant parts and preparation methods of use are consistent with the records in ancient texts. Almost 300 phytochemicals have been identified from Bletilla, and some of them possess important pharmacological activities, which support its traditional uses and suggest the important medicinal development potential of this genus. This review has demonstrated that Bletilla, as an important medicinal plant of Orchidaceae, still requires further research to fathom its medicinal potential.

    For instance, it is necessary to enhance the quality control procedures based on the chemical components and pharmacological activity of Bletilla. The chemical composition and pharmacological properties of Bletilla are critical areas of current research. According to previous studies, the main bioactive components of Bletilla can vary greatly according to its origin, harvest time, distribution, storage, and adulteration. However, variation in bioactivities caused by the differences in Bletilla constituentshave not been explored extensively yet. To develop clinical applications of Bletilla, it is crucial to further explore the mechanism of action between its chemical composition variation and its pharmacological actions.

    In addition, although the tuber has historically been the main medicinal part of Bletilla, research has shown that the chemical composition in other parts of Bletilla, such as stems, leaves, and flowers, also give these parts a variety of pharmacological activities. Further in-depth analysis of the chemical components and pharmacological activities of different parts of this genus is worthwhile, to explore the specific chemical basis of its pharmacological activities, develop related drugs, and promote clinical applications. For example, Bletilla polysaccharide has good hemostasis and astringent wound effects[110], so it may have the potential to be developed into a drug or related medical materials to stop bleeding and heal wounds.

    Finally, as a cautionary note, many unrestrained collections and the destruction of habitats have made the resources of wild Bletilla rarer. In addition to protecting the wild populations of Bletilla, appropriate breeding techniques should be adopted to meet the commercial needs of this economically important genus, thereby allowing its sustainable use in commerce.

    The authors confirm contribution to the paper as follows: study conception and design, funding acquirement: Long C; data analysis, draft manuscript preparation, literature review: Fan Y, Zhao J; manuscript revise and language editing: Wang M, Kennelly EJ, Long C. All authors reviewed the results and approved the final version of the manuscript.

    The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher. Requests to access these datasets should be directed to Yanxiao Fan (fanyanxiao0510@163.com).

    This research was funded by the Yunnan Provincial Science and Technology Talent and Platform Plan (202305AF150121), Assessment of Edible & Medicinal Plant Diversity and Associated Traditional Knowledge in Gaoligong Mountains (GBP-2022-01), the National Natural Science Foundation of China (32370407, 31761143001 & 31870316), China Scholarship Council (202206390021), and the Minzu University of China (2020MDJC03, 2022ZDPY10 & 2023GJAQ09).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Hooks T, Masabni J, Sun L, Niu G. 2022. Effects of organic fertilizer with or without a microbial inoculant on the growth and quality of lettuce in an NFT hydroponic system. Technology in Horticulture 2:1 doi: 10.48130/TIH-2022-0001
    Hooks T, Masabni J, Sun L, Niu G. 2022. Effects of organic fertilizer with or without a microbial inoculant on the growth and quality of lettuce in an NFT hydroponic system. Technology in Horticulture 2:1 doi: 10.48130/TIH-2022-0001

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Effects of organic fertilizer with or without a microbial inoculant on the growth and quality of lettuce in an NFT hydroponic system

Technology in Horticulture  2 Article number: 1  (2022)  |  Cite this article

Abstract: Organic food continues to increase in popularity worldwide. Similarly, hydroponic production of leafy greens is expanding globally and is an important component of the world's food supply. The purpose of this study was to evaluate the growth and quality of lettuce using six nutrient film technique (NFT) hydroponic systems. There were three treatments: organic fertilizer with or without a microbial inoculant and a conventional inorganic fertilizer as a control. The experiment was repeated over time. Results showed that the plants grown with organic fertilizer with additional microbial inoculant achieved similar shoot fresh and dry weight to those of the control, and dry weight was 17% higher than the organic fertilizer without inoculant. Nitrogen content in the shoot tissue of plants treated with organic fertilizer with inoculant was 10% and 24% greater than the control and the organic fertilizer without inoculant, respectively. However, when the organic fertilizer with inoculant was reused in a second experiment, shoot fresh and dry weight of plants in organic fertilizer with inoculant was lower than those in the control but were still higher compared to the organic fertilizer without inoculant. Additionally, electrical conductivity (EC) and pH of the organic fertilizer solutions fluctuated widely. Interestingly, relative chlorophyll content measured as SPAD and anthocyanin content in the leaf tissue increased in plants treated with organic fertilizer, regardless of inoculant, by 19% and 9%, respectively.

    • Demand for organic produce and products continues to increase worldwide. The organic market was valued at more than 42billionintheUnitedStatesandmorethan97 billion globally in 2017[1]. Sales of organic crops in the U.S. have increased by 38% since 2016, and organic leafy greens, including lettuce and spinach, account for nearly $600 million in annual sales[2]. Organic agriculture is based on principles of sustainability and conservation of the natural environment. Although modern farming technologies can be utilized, the use of synthetic substances are prohibited, such as inorganic fertilizers or chemical pesticides. Because of this, organic farming must rely on natural substances for managing pests, and organic based fertilizers such as manure or compost. However, this can result in lower productivity (i.e. yield) compared to conventional farming. Many studies have been published on the yield gap between organic and conventional agriculture, and meta-analysis of the data indicates a yield gap of 19%[3]. However, this gap is highly contextual. Depending on the management practice and type of crops, organic yields can be comparable to conventional yields[4]. Therefore, there is need for data driven information and application of organic materials and practices for effective and productive organic agriculture.

      In the 2017 fall meeting, the National Organic Standard Board (NOSB) passed a motion to allow hydroponically grown crops be eligible for certification[5]. Although organic hydroponic production has been debated due to its soilless nature[6], this decision will inevitably raise interest and allow U.S. growers to produce certified organic produce using organic culture in hydroponic systems. However, there has been very little research-based information on organic culture for hydroponics. This is partly because organic certification of hydroponically grown crops are prohibited in most European countries[7]. Nevertheless, there is a need for scientific research and quantitative data on organic culture in hydroponic systems to guide and support this burgeoning industry.

      Greenhouse hydroponic production of leafy greens is expanding globally and is an important component of the world's food supply[8]. Hydroponics is an excellent method to produce leafy greens due to its soilless nature and ability to recirculate water and nutrients. For example, lettuce production in hydroponics can increase yield tenfold while using 90% less water compared to traditional field agriculture[9]. Because of this, hydroponics is commonly used in controlled environment agriculture (CEA) which includes greenhouses, high tunnels, and indoor plant factories[10]. CEA is expected to play a critical role in reinforcing food security in urban areas[11]. Despite these positive aspects, hydroponic production relies largely on water-soluble inorganic fertilizers and conventional substrates. However, there is potential to combine the productiveness of hydroponics with organic fertilizers and practices.

      Although organic hydroponic production is possible, there is limited research available on the topic. Williams and Nelson[12] grew organic lettuce in a nutrient film technique (NFT) hydroponic system but noted challenges in organic fertilizer management compared to conventional. Shinohara et al.[13] reported the successful use of microorganisms to mineralize the organic fertilizer into available forms of nutrients essential for plant growth. Therefore, the addition of microorganisms to mineralize the organic fertilizer might be critical for effective organic hydroponic production. However, there is limited information on the process of mineralization and even less quantitative data on its effectiveness in organic hydroponics. For example, Saijai et al.[14] stated that pH was one of most important factors that influenced the rate of mineralization, but other factors can contribute as well, such as the makeup of the organic fertilizer.

      There are many commercially available products for organic hydroponic production, including liquid organic fertilizers that are made from a variety of materials derived from plant and animal sources, including fish and seaweed emulsions, fish hydrolysates, and oilseed extract. There are also many challenges in using liquid formulations of organic fertilizers, namely, clogged tubing in the delivery system, biofilm formation, low nitrogen availability, and possibly high content of unwanted minerals such as sodium[12,15]. Another challenge is that some organic fertilizers contain beneficial microbes while others do not. To make matters more complicated, many products do not have accurate or detailed listing of the compositions and/or concentrations of the nutrients due to proprietary reasons.

      Microbial inoculants are beneficiary microorganisms applied to either soil or plant to improve productivity and crop health through enhanced mineralization of organic fertilizers and nutrient availability. Low nutrient availability is one of the primary causes of low yield in organic culture. Therefore, the purpose of this study was to evaluate the performance of lettuce in a hydroponic system using a commercially available certified organic liquid fertilizer compared to a conventional inorganic fertilizer. Additionally, a commercially available microbial inoculant was used to evaluate the mineralization process and quantify its effectiveness on the growth and quality of lettuce in a hydroponic system.

    • Lettuce seed (Lactuca sativa L. 'Red Mist', Osborne Seed Company, Mount Vernon, WA), was sown in 25-mm coconut coir plugs (Riococo, Irving, TX). Both seed and substrate were certified USDA Organic. Plugs were soaked in reverse osmosis (RO) water until fully expanded and then placed in 128-cell trays. A single seed was sown per plug and trays were placed in an indoor propagation rack. Plastic domes were used to cover the trays to maintain high relative humidity during germination. A heating mat was placed underneath the tray to warm the substrate to 24 °C. After germination, domes were removed and two broad band full spectrum LED light bars (Illumitex, Austin, TX) with a photosynthetic photon flux density (PPFD) of 150 µmol m−2 s−1 were turned on for a 16-h photoperiod. Two fans measuring 10 cm in diameter were used to provide air circulation in the propagation shelf while the lights were on. Seedlings were sub-irrigated daily with half-strength nutrient solution at an electrical conductivity (EC) of 1.0 mS cm−1 and a pH of 5.6. When seedlings reached sufficient growth with two expanded true leaves, they were moved to a greenhouse to harden-off before transplanting. At approximately 14 days after sowing (DAS), uniform seedlings were transplanted to six nutrient film technique (NFT) hydroponic systems (Cropking, Lodi, OH).

    • There was a total of six independent NFT hydroponic systems, each with four channels measuring 2.4 m with 12 holes (2.5 cm diameter) for a total of 48 plants per system. Each system had a 94.6-L reservoir tank with a submersible pump (1,512 L h−1) that pumped the nutrient solution to the channels via polytube plumbing and drip lines. There was a single drip line per channel with a flow rate of 39 L h−1. The channels were positioned on an aluminum frame with a decline of 3% from the inlet to the outlet of the nutrient solution to provide constant flow of a thin film of solution inside the channel. The solution drained out the outlet end of the channel into a manifold that recirculated the solution to the reservoir tank.

    • Two replicated greenhouse experiments were conducted at the Texas A&M AgriLife Research Center in Dallas, TX (32°59'13.2"N, 96°45'59.8"W; elevation 131 m) from 29 May to 12 June 2020 (Experiment 1) and 24 June to 09 July 2020 (Experiment 2). The greenhouse temperature was controlled by a heating, ventilation, and air conditioning (HVAC) system equipped in the adjacent office building. Throughout each experiment, greenhouse air temperature and photosynthetic active radiation (PAR) were recorded by a datalogger (Campbell Scientific Inc., Logan, UT). The actual daily average air temperature and daily light integral (DLI) for each experiment are presented in Fig. 1. The greenhouse was divided into two blocks to account for environmental differences between the east and west sides. Due to low DLI recorded prior to the start of the first experiment, supplemental light was provided by LED light fixtures (LumiGrow, Emeryville, CA) with a spectrum of blue, green, and red light (23%, 4%, and 73%, respectively) and a photosynthetic photon flux density (PPFD) of 143 µmol m−2 s−1 measured at plant height, to achieve a 16-h photoperiod throughout the treatment duration in Experiment 1. However, no supplemental lighting was provided in Experiment 2.

      Figure 1. 

      Average daily air temperature and daily light integral (DLI) in the greenhouse throughout the two experiments. The duration of both experiments was 14 days after transplanting (DAT). Experiments 1 and 2 were conducted from 29 May to 12 June and from 24 June to 09 July 2020, respectively.

    • A total of three treatments were used, including a control and two organic treatments (organic fertilizer alone, organic fertilizer with microbial inoculant). The control consisted of a custom conventional nutrient solution blend (modified Hoagland formula) and was prepared using tap water and fertilizer salts at a mM rate of 10.7 N, 1.13 P, 5.38 K, 3.25 Ca, 1.44 Mg, and 1.44 S, and a μM rate of 53.7 Fe, 27.8 B, 6.0 Mn, 1.83 Zn, 1.10 Cu, and 0.63 Mo. The following fertilizer salts were used to prepare the nutrient solution: potassium nitrate, potassium phosphate, calcium nitrate, magnesium sulfate, iron chelate, boric acid, manganese sulfate, zinc sulfate, copper sulfate, and molybdic acid. The control solution had an EC of 1.8 mS cm−1. In the second experiment, the control was prepared at a slightly lower rate with an EC of 1.6 mS cm−1 to better match the EC of the organic treatments. The organic fertilizer treatment was prepared using tap water and a liquid organic fertilizer (Pre-Empt, Coastal Fertilizer & Supply Inc., Labelle, FL) at the recommended label rate of 10 mL L−1. According to the product label, Pre-Empt is a fermented colloid molasses with a unique microbial complex; however, no quantitative information for nitrogen, phosphorus, and potassium is indicated. The following nutrient elements were included in the package with guaranteed analysis (mM): calcium (5.00), magnesium (8.33), iron (1.79), manganese (1.82), zinc (1.54), and boron (1.85). The other organic treatment (organic with inoculant) was prepared by adding a microbial inoculant (TerraBella, Aquabella Organic Solutions, Sebastopol, CA) at a rate of 50 mg L−1 to the above organic treatment. While no specific species of microorganisms is given, the TerraBella product label indicated the following information: 80 million colony forming units (CFUs, units/mL) of aerobic bacteria; 170 CFUs of anaerobic bacteria. The inoculant, containing a proprietary blend of beneficial plant growth-promoting rhizobacteria, was activated according to the label at a rate of 10 mL L−1 in reverse osmosis (RO) water 48 h prior to its addition to the fertilizer. Previous trials (unpublished) showed low magnesium rates of 10 mg L−1; therefore, an organic magnesium supplement was added in the first experiment at the rate of 1 mL L−1 which provided approximately 1.25 mM of additional Mg. The solutions of the organic fertilizer were reused in the second experiment to evaluate the effectiveness of the microbial inoculant across both experiments. For the second experiment, the organic treatments were topped off with tap water and replenished with organic fertilizer to an EC of 1.6 mS cm−1. For the organic fertilizer with inoculant treatment, the solution was re-inoculated at the same rate as the first experiment. Both Pre-Empt organic fertilizer and TerraBella microbial inoculant are certified organic products.

      The pH of the control solution was adjusted to 5.6 at the start of both experiments. For the organic treatments, pH was adjusted to approximately 6.0 at the start of both experiments using phosphoric acid for decreasing pH and a mixture of potassium hydroxide and potassium carbonate for increasing pH. Treatments were initiated after seedlings were transplanted to the hydroponic systems on the 29th of May for the first experiment and 24th of June for the second experiment. For both experiments, treatments lasted two weeks.

    • Both experiments were arranged as a randomized complete block design (RCBD) with two blocks. Only two blocks were used due to limitations in space and equipment (NFT systems). The three treatments were randomized per block with a total of 288 plants or 48 plants per block. All response variables were analyzed using ANOVA with JMP 14.2 (SAS, Cary, NC). Mean separations were analyzed using Tukey's Honest Significant Difference (HSD) test and pair-wise differences were analyzed using Student's t-Test at an alpha of 0.05. Experimental differences were tested in the plant growth response variables and were determined to be significant (P ≤ 0.007), therefore the experimental data and results were analyzed and presented separately.

    • Throughout both experiments, the treatment solutions in the reservoirs were monitored daily for EC, pH, and solution volume. The EC and pH were measured using a combo meter (Bluelab, Tauranga, New Zealand). Volume was estimated using a custom ruler with 9.5-L increments. On a weekly basis, expanded true leaves were counted and the natural plant height (h) and two perpendicular widths (w) were measured by hand using a ruler on nine plants per treatment. A growth index (GI) was calculated as follows[16]:

      (h+¯w)2

      where h is the natural plant height, and ¯w is the average of the two perpendicular widths.

      Experiments were terminated when the adjacent plants were touching each other and leaves started overlapping; thus, plants were not fully mature compared to field lettuce standard but they were larger than baby lettuce. At termination of the experiments, relative chlorophyll content, shoot and root fresh weight (FW), and dry weight (DW) of nine plants were collected. The relative chlorophyll content was taken from the average of three mature leaves per plant, nine plants per treatment (the same plants for GI measurement) using a portable SPAD-502Plus meter (Konica Minolta, Chiyoda, Japan). The tissue DW was measured following complete dryness in a Heratherm OGS750 drying oven (Thermo Fisher Scientific, Waltham, MA) at 70 °C. Shoot tissue samples were ground in a Wiley mill (Thomas Scientific) and sent for elemental analysis at the Soil and Water Testing Laboratory (College Station, TX). In the second experiment, leaf tissue samples were collected for phytonutrient analysis from representative mature leaves of three plants. In brief, fresh tissue samples (1 g) were collected and immediately frozen in liquid nitrogen and stored in a freezer at −80 °C. Subsequently, these samples were ground in a mortar and pestle using liquid nitrogen and extracted in methanol. The extracted samples were then analyzed for anthocyanins and total phenolic compounds (TPC) using the methods described by Silva et al.[17] and Ainsworth and Gillespie[18], respectively. Anthocyanins were measured using a Genesys UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and calculated according to the following formula:

      V×n×M×A×100ε×m

      where, V: The volume of extraction liquid (ml), n: Dilution factor, M: Molecular weight of cyanidine-3-glucoside (449.2 g), A: Absorbance @ 530 nm, ε: molar extinction coefficient (29,600), and m: weight of sample. TPC was determined using the Folin & Ciocalteu's reagent, measured using a microplate spectrophotometer (BioTek, Winooski, VT, USA) at 765 nm, and reported as mg of Gallic Acid Equivalents (GAE) divided by g of FW.

    • Results from Experiment 1 indicated that plant growth index (GI) was lower in the organic nutrient solution compared to the control (Fig. 2). However, the addition of the inoculant in the organic solution increased GI by 9% and was comparable to the inorganic control. In Experiment 2, a similar trend was observed whereby plant GI increased by 9% with the addition of the inoculant to the organic nutrient solution. However, the control resulted in the greatest plant GI, which was approximately 11% and 21% greater than the organic solutions with or without the inoculant, respectively.

      Figure 2. 

      Growth Index (GI) of the lettuce variety 'Red Mist' grown for two weeks in an NFT hydroponic system fertilized with a conventional (Control) or an organic fertilizer with or without a microbial inoculant. The experiment was replicated and presented separately: Experiment 1 (a) and Experiment 2 (b). Bars represent standard error. Means followed by different letters indicate significant differences among treatments according to Tukey's HSD test (P < 0.05).

      In Experiment 1, shoot fresh weight (FW) showed a similar trend as plant GI; however, no significant differences were observed among the treatments (Fig. 3a). The addition of the inoculant increased shoot dry weight (DW) by 17% and was comparable to the control. These results were supported by the plant GI data, which was strongly correlated (r2 = 0.62). There were no significant differences among treatments in shoot water content (WC) and no apparent trends observed in the data.

      Figure 3. 

      Shoot fresh weight (FW), dry weight (DW), and water content (WC) of the lettuce variety 'Red Mist' (per plant) grown for two weeks in an NFT hydroponic system fertilized with a conventional (Control) or an organic fertilizer with or without a microbial inoculant. The experiment was replicated and presented separately: Experiment 1 (a) and Experiment 2 (b). Bars represent standard error. Means followed by different letters indicate significant differences among treatments according to Tukey's HSD test (P < 0.05).

      In Experiment 2, shoot FW was reduced by 31% and 41% in the organic treatments with or without inoculant, respectively, compared to the control (Fig. 3b). These were large reductions in FW that were not observed in Experiment 1. Additionally, the inoculant did not appear to affect FW. However, there were differences in DW, similar to Experiment 1. Specifically, there was an increase in DW by 24% in the organic nutrient solution with inoculant compared to without. However, even with the inoculant, the DW in the organic treatment was not comparable to the control, which was 19% greater. Again, these results were supported by the plant GI data, which was strongly correlated (r2 = 0.93). In contrast to Experiment 1, shoot WC indicated a greater proportion of dry biomass in the organic treatments compared to the control. Specifically, the shoot WC was 1.5% and 1.7% less in the organic solution with or without inoculant, respectively.

    • The relative chlorophyll content (SPAD) increased in the organic treatments compared to the control in both experiments (Fig. 4). In Experiment 1, SPAD increased by 18% and 26% in the organic treatments with or without inoculant, respectively. In Experiment 2, SPAD increased by 19% and 13%, respectively. Although there were significant differences between the two organic treatments in both experiments, it remained unclear whether the inoculant had an overall positive effect on SPAD.

      Figure 4. 

      Relative Chlorophyll Content (SPAD) of the lettuce variety 'Red Mist' grown for two weeks in an NFT hydroponic system fertilized with a conventional (Control) or an organic fertilizer with or without a microbial inoculant. The experiment was replicated and presented separately: Experiment 1 (a) and Experiment 2 (b). Bars represent standard error. Means followed by different letters indicate significant differences among treatments according to Tukey's HSD test (P < 0.05).

      Results of the fresh tissue extraction and analysis showed that plant anthocyanin content tended to increase in plants treated with organic nutrient solution with inoculant compared to plants treated with conventional solution (Fig. 5). Specifically, anthocyanin in the shoot tissue increased by 9% in the organic solution with inoculant compared to the inorganic control. Regarding total phenolic content (TPC), there were no significant differences among treatments and no clear trend in the data.

      Figure 5. 

      Anthocyanin and total phenolic content (TPC) of the lettuce variety 'Red Mist' grown for two weeks in an NFT hydroponic system fertilized with a conventional (Control) or an organic fertilizer with or without a microbial inoculant. The experiment was replicated but only data from the second experiment is presented. Bars represent standard error. Means followed by different letters indicate significant differences among treatments according to Tukey's HSD test (P < 0.05).

    • In Experiment 1, the leaf tissue content of macronutrients was generally greatest in plants treated with the organic nutrient solution with inoculant (Table 1). The concentration of N was 10% and 24% greater than the control and organic treatment without inoculant, respectively; P was 12% and 29% greater; Mg was 11% and 13% greater; S was 19% and 41% greater; and Fe was 14% and 17% greater. The leaf tissue mineral content of Mn and Zn were greater in plants treated with the organic solution with inoculant compared to the organic solution without inoculant, but not the control. In this case, Mn and Zn were 113% and 25% greater, respectively. There were no significant differences among the treatments for the leaf tissue mineral content of K, Ca, B, and Cu.

      Table 1.  Leaf tissue mineral concentration of the lettuce variety 'Red Mist' grown for two weeks in an NFT hydroponic system fertilized with a conventional (Control) or an organic fertilizer with or without a microbial inoculant.

      ElementControlOrganicOrganic with
      inoculant
      Experiment 1
      (mg/gDW)
      N59.85 b52.99 c65.94 a
      P9.04 b7.83 c10.09 a
      K80.7967.9753.09
      Ca7.938.585.64
      Mg3.42 b3.36 b3.80 a
      S4.04 b3.41 b4.80 a
      Fe0.102 b0.099 b0.116 a
      B0.0470.0380.063
      Cu0.0160.0150.025
      Mn0.131 ab0.102 b0.217 a
      Zn0.064 ab0.056 b0.070 a
      Experiment 2
      N49.4553.3447.46
      P7.407.457.32
      K68.36 a46.08 b43.92 b
      Ca9.65 a6.40 b4.87 c
      Mg3.362.893.12
      S2.95 b3.68 a4.07 a
      Fe0.079 b0.099 a0.086 b
      B0.019 b0.043 a0.046 a
      Cu0.008 b0.017 a0.020 a
      Mn0.066 b0.232 a0.247 a
      Zn0.047 ab0.054 a0.039 b
      The experiment was replicated, and data is presented from both experi-ments. Means followed by different letters indicate significant differences among treatments according to Tukey's HSD test (P <0.05).

      In Experiment 2, the leaf tissue content of micronutrients was generally greatest in plants treated with the organic nutrient solution, regardless of inoculant, compared to the conventional (Table 1). The concentration of B and Cu were more than double that of the control, while Mn was more than threefold in the organic treatments. Additionally, the macronutrient S was 38% and 25% greater in the organic treatment with or without inoculant, respectively. However, the macronutrients K and Ca were greater in the control compared to the organic treatments. Specifically, K was 56% and 48% greater than the organic solution with or without inoculant, respectively, and Ca was 98% and 51% greater, respectively. There was a similar trend in the first experiment, although there were no significant differences. Finally, there were no significant differences among the treatments for the minerals N, P, and Mg.

    • In Experiment 1, the EC of the control solution remained stable, but steadily increased in the organic treatments throughout the duration of the study (Fig. 6). By the end of Experiment 1, the EC of the organic treatments was 2.5 mS cm−1 which was an increase of 32% compared to the control. Overall, the average EC of the control, organic, and organic with inoculant was 1.9 ± 0.0, 2.1 ± 0.2, and 2.1 ± 0.2 mS cm−1, respectively. Concurrently, the volume of the solutions decreased throughout the experiment by 46%, 36%, and 41%, respectively (data not presented).

      Figure 6. 

      Electrical conductivity (EC) and pH of the three nutrient solutions, including a conventional (Control) and an organic fertilizer with or without inoculant, used to grow lettuce in an NFT hydroponic system in a greenhouse for two weeks, or 14 days after transplant (DAT). The experiment was replicated twice. The control solution was prepared fresh for both experiments while the organic solutions were replenished and re-used in Experiment 2 to evaluate the effectiveness of the microbial inoculant.

      The pH of the control solution remained relatively stable while the organic solutions increased notably during the first week, but then tapered off toward the end of the experiment (Fig. 6). The pH of the organic solutions with or without inoculant reached a maximum of 7.4 and 7.3, which represented an increase of 20% and 23%, respectively, compared to the start of the experiment. Overall, the average pH in the control, organic, and organic with inoculant nutrient solutions was 5.9 ± 0.2, 6.9 ± 0.3, and 7.0 ± 0.4, respectively.

      In Experiment 2, the EC of all the nutrient solutions tended to remain stable throughout (Fig. 6), which was dissimilar compared to the Experiment 1 for the organic treatments. Overall, the average EC of the control, organic, and organic with inoculant was 1.7 ± 0.0, 1.7 ± 0.1, and 1.7 ± 0.0 mS cm−1, respectively. Concurrently, the volume of the solutions decreased throughout the experiment by 29%, 20%, and 22% respectively (data not presented).

      In Experiment 2, the pH of the control solution was relatively stable compared to the organic solutions, which was similar to Experiment 1 (Fig. 6). However, the organic treatments behaved dissimilarly to the first experiment. Specifically, the pH of the organic solution increased slowly during the first week to 6.8, and then decreased slowly during the second week to 5.6. In contrast, the organic solution with inoculant barely increased to a pH of 6.5, followed by a more rapid decrease to a minimum of 4.8 at the end of the experiment. Overall, the average pH in the control, organic, and organic with inoculant nutrient solutions was 5.6 ± 0.2, 6.2 ± 0.6, and 5.7 ± 0.6, respectively.

    • Our results showed that plant growth with organic fertilizer and inoculant was equal to the conventional fertilizer in the first experiment, based on growth index, and fresh and dry weight. These results indicate that hydroponic production of lettuce using organic fertilizer with a microbial inoculant is viable and has the potential to achieve similar yields to conventional fertilizer. For the organic fertilizer treatments, we did not observe blockage of tubes, but we noticed that the continuous nutrient supply caused a problem in the root zone of the propagation plugs, where roots did not develop well due to low dissolved oxygen as observed in our previous trials, due to the formation of biofilm inside the propagation plugs. After we changed the nutrient supply to 15 min per hour, giving the roots a 'dry' period, crop performance improved.

      Shinohara et al.[13] reported an increase in fresh and dry weight of butterhead lettuce using an organic fertilizer with a microbial inoculant in a custom hydroponic system, compared to conventional fertilizer. However, the results from our second experiment showed that plant growth decreased when treated with organic fertilizer compared to the conventional control, but the addition of inoculant increased growth in the organic treatment. A possible reason for the reduced growth is insufficient nutrient availability in the organic treatments as evidenced in the lower tissue K and Ca content, which may be due to slower rate of mineralization in the organic treatments that did not meet the plant growth rate. Saijai et al.[14] reported that the rate of mineralization was highest in a nutrient solution with a pH of 7.5. Our results appear to agree, since the pH of the organic fertilizer solutions with or without inoculant reached a maximum of 7.3 in the first experiment but decreased to a minimum of 4.8 and 5.6, respectively, in the second experiment. This is most likely attributed to poor mineralization (and subsequently nutrient availability) in the second experiment.

      Both experiments showed that the addition of a microbial inoculant improved plant growth compared to the organic fertilizer applied alone. The inoculant provides the necessary microorganisms to mineralize larger organic compounds into smaller inorganic nutrients essential for plant growth. Therefore, it is presumed that there was less nutrient availability in the organic fertilizer solution without inoculant, which was evidenced by the reduced mineral content in the shoot tissue of the plants. Moreover, possible phytotoxic effects could have inhibited plant growth. Garland et al.[19] reported phytotoxic effects from soluble organic compounds in a recirculating hydroponic system, but then remediated the effects through microbial activity. Lee et al.[20] identified several organic acids that accumulated when hydroponic nutrient solution was recycled. Asao et al.[21] mitigated the toxicity of an organic acid with a microbial strain and improved the yield of cucumber in a hydroponic system. Our results align with these studies in that the addition of a microbial inoculant benefited plant growth in a hydroponic system.

      The higher SPAD readings in the organic treatments indicate greater relative chlorophyll and nitrogen content in the leaves[22]. A higher SPAD reading can also indicate thicker leaves with higher contents of phytonutrients[23]. However, this was not supported by the plant growth (both FW and DW) and the leaf N concentration in our study. These results may indicate that SPAD readings may have a poor correlation with leaf N concentration in organically grown plants. While not quantified, leaf color in organic treatment appeared darker compared to that in the control. Plant quality increased with the organic fertilizer and inoculant compared to the conventional fertilizer, based on anthocyanin results. Anthocyanins are blue/purple pigments in plants and have been shown to have antioxidant and anti-inflammatory properties[24,25]. Overall, these results indicate that there is potential for organic hydroponic production to produce lettuce with improved quality to conventional methods.

      In the first experiment, the mineral content of both macro and micronutrients was highest in the shoot tissue of lettuce plants treated with the organic fertilizer and inoculant. This indicates that sufficient mineralization was occurring in the solution, and essential nutrients were available for plant uptake. This is important for hydroponic production of crops, where a continuous supply of nitrogen is critical for optimum plant growth[26]. For organic fertilizer, the mineralization process is necessary to produce ammonium and subsequently nitrate (via ammonification and nitrification, respectively), the latter of which is the preferred source of nitrogen for plants[27]. However, it is important to note that high nitrate content in edible leafy greens is undesirable due to human health concerns[28]. In the second experiment, the nitrogen levels in the shoot tissue were less than those observed in the first experiment and there were no differences among treatments, indicating similar nitrate levels.

      Additionally, calcium and potassium concentrations in the shoot tissue were lower than those of the control, indicating these macronutrients might be limited in organic liquid fertilizer for hydroponic production. It may be possible to increase Ca and K by supplementing with specific Ca and K organic fertilizers, similar to what was done with Mg in this study. That is, multiple organic fertilizers may be necessary to provide a 'full spectrum' of essential macronutrients.

      Regarding the EC and pH of the nutrient solutions, our results indicated that organic fertilizer is more dynamic than conventional fertilizer throughout the growing cycle of lettuce. The steady increase in the solution EC of the organic treatments in the first experiment could be explained by the mineralization process, which can increase availability of ions in the solution. This would also explain why the EC in the control solution did not increase throughout the experiment, since the conventional fertilizer is already in an inorganic form, therefore no mineralization could occur. However, the depletion of the reservoir solution through evapotranspiration would outpace the differential uptake of ions by the plants, as shown by Niu et al.[29], which can lead to a more concentrated nutrient solution over time. In our case, less than half of the reservoir solution was depleted by the end of the experiment, therefore this impact was less noticeable.

      In the second experiment, the EC was more stable for all the treatment solutions. This was attributed to the slightly lower average EC of all the treatments at the start of the second experiment compared to the start of the first (1.6 and 1.8 dS m−1, respectively). Additionally, since the organic solutions were re-used, lower rates of mineralization were attributed to the static nature of the EC in the second experiment. At the start of the second experiment, approximately 25% of the original amount of liquid organic fertilizer was used to replenish the solutions to the desired EC level. Williams and Nelson[12] reported challenges in using EC to indicate nutrient levels in an organic fertilizer to grow lettuce (Lactuca sativa L. var. 'Rex') in an NFT hydroponic system.

      Managing pH in a hydroponic system is important to prevent certain nutrients from forming precipitates and becoming unavailable for plant uptake. A pH range of 5.8−6.4 is recommended for most plants in a hydroponic system[30]. For organic fertilizer, this may not be possible due to large fluctuations, as our results indicated. Over the course of both experiments, the pH of the organic solutions fluctuated from a high of 7.3 to a low of 4.8. Williams and Nelson[12] also reported large pH fluctuations and difficulty in managing the pH of an organic nutrient solution. These large changes in pH might be attributed to the mineralization of the organic fertilizer in conjunction with the uptake of available nutrients by the plants. For example, Imas et al.[31] showed that pH increases in solution when nitrate (NO3) is taken up by plants due to simultaneous H+ uptake in order to balance charge. Similarly, pH decreases when ammonium (NH+4) is taken up by plants due to simultaneous H+ efflux. Based on this, the pH fluctuations observed in our study indicate that more nitrification and nitrate uptake was occurring in the first experiment, and more ammonification and ammonium uptake was occurring in the second experiment. More research is needed on the management of pH in an organic fertilizer solution with a microbial inoculant in order to better understand the mineralization process for effective organic hydroponic production.

    • Our study showed that hydroponic production with organic fertilizer is feasible and can produce lettuce with yields similar to conventional fertilizer. Additionally, our results emphasize the importance of a microbial inoculant in conjunction with an organic fertilizer for more effective mineralization and enhanced plant growth. There is also potential for organic hydroponic production to produce high quality crops with increased pigmentation and phytonutrient content. However, managing organic fertilizer solution with inoculant over multiple growing cycles can be challenging and supplemental organic fertilizers with different macro nutrients such as Ca, K and Mg may be needed to match the need for optimal plant growth. The EC and pH of the organic fertilizer solution can fluctuate widely, and that a high pH of approximately 7.0 may be more effective for mineralization and plant growth. More research is needed to better understand the mineralization process of organic fertilizer in a nutrient solution for effective organic hydroponic production.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2022 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (1) References (31)
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    Hooks T, Masabni J, Sun L, Niu G. 2022. Effects of organic fertilizer with or without a microbial inoculant on the growth and quality of lettuce in an NFT hydroponic system. Technology in Horticulture 2:1 doi: 10.48130/TIH-2022-0001
    Hooks T, Masabni J, Sun L, Niu G. 2022. Effects of organic fertilizer with or without a microbial inoculant on the growth and quality of lettuce in an NFT hydroponic system. Technology in Horticulture 2:1 doi: 10.48130/TIH-2022-0001

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