Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa

*Kenneth Yongabi Anchang and Henrik Toft Simonsen*

#### **Abstract**

The work described here covers an examination of new bioproducts based on sub-Saharan bryophytes. The work includes in vitro testing of extracts from moss and liverworts against plant pathogenic microbes causing food decay and field crop losses. Additionally, we have shown specific antimicrobial activities of *Marchantia debilis* and **moss** against *Erwinia spp* and *Pseudomonas spp*. The extracts were also tested against aflatoxin-producing fungi isolated from food crops such as maize and peanuts. The efficacy of the extracts on clinical dermatological fungal isolates like *Dermatophilus congolensis* has not been reported. This led to the production of an antifungal solution of bryophyte extracts, which was tested in vivo on animals with skin diseases caused by *Dermatophilosis*. Around 99.5% of the animals were treated. The antifungal solution for treatments has been labeled **Bryosol**, while the disinfectants solution is labeled Bryo-disinfectants and the crop-fungicide is labeled Bryo-fungicides. A mini field pilot trial with Bryo-fungicide showed that crops infected with pathogenic fungi were treated. The results provide the first attempt to demonstrate the use of bioproducts for organic treatment of agricultural crops and diseases in animals based on sub-Saharan bryophytes.

**Keywords:** moss, Marchantia, Bryosol, disinfectants, antifungal, bioproducts, foods, *Dermatophilosis*

#### **1. Introduction**

Bryophytes are non-vascular plants, which are constituted of mosses, liverworts, and hornworts [1]. Although not usually seen to have any importance, bryophytes have recently been used as bioindicators of pollution and are often used for decorations. However, the medicinal value of bryophytes is huge with a panoply of bioactive compounds isolated from bryophytes, especially liverworts [1, 2]. Bioactive compounds have been isolated from liverworts from Asia, Europe, and South Africa. For example, Allison in 1975 identified a number of bioactive compounds from liverworts in New Zealand. Volatile constituents have been identified in liverworts like *Tritomaria polita*, *Marsupella emarginata*, *M. aquatic*, and *M. alpine* [3].

It has been seen that bryophytes are rich in diverse phytochemicals such as sesquiterpenoids, norsesquiterpenoids, anthocyanidins, riccionidins etc. with interesting biological activities, such as antimicrobial, antifungal, insect-repellent, molluscicidal, cardiotonic activity, and fragrance compounds among others [1]. Bryophytes

are very common across the world, particularly in wet areas like Cameroon. The ecology of Cameroon is rich in algae and lichens; bryophytes in Cameroon are part of the Congo forest and the highlands from Mount Cameroon via the Atlantika Mountains to the Mandara Mountains collectively constituting the Congo forest from Nigeria, ranges from 1400 to 4000 m, and harbors a rich biodiversity of both lower and higher plants. A survey of bryophytes in Cameroon revealed many unidentified species with familiar dormant species such as *Marchantia spp* [2].

Phenanthrenes and other phenolics have been isolated from in vitro cultures of *Marchantia polymorpha.* Recently, extensive report was published on the biology and constituents and chemistry and organic natural products of bryophytes [1, 4], though this lacks data on bryophytes from West and Central Africa, especially Cameroon. Screening of bryophytes and lower plants for biologically active compounds from Cameroon is of great importance considering that Cameroon is centrally placed in Africa and harbors all the ecological and geographical characteristics widespread across Africa. The search for bioactive compounds from plants in the past 30 years in Africa has concentrated on higher plants with little or no interest on bryophytes, which is the same in the rest of the world [2]. The prevalence of many plant and animal pathogenic diseases is growing along with drug resistance strains. This generates huge losses in agricultural yield and productivity across Africa. Treatment and management are expensive for many African farmers and therefore a cheap alternative, preferably organic, is needed [3].

Bioactive compounds from bryophytes could bridge this gap. Here, we show that new drug leads could be identified from bryophytes from Cameroon to address plant pathogenic diseases and animal diseases like *Dermatophilosis* infection in cattle.

#### **2. Dermatophilosis in animals**

Dermatophilosis is caused by the bacterium *Dermatophilus congolensis*, which is an aerobic actinomycete (facultatively anaerobic) and usually affects animals and occasionally humans [5].

Dermatophilosis is distributed worldwide, prevailing in tropical areas, and related to humid environments and other factors, such as poor veterinary services, coinfection with a number of bacterial infections, especially in animals with compromised immune systems, and poor hygiene conditions in favor of its occurrence and spread.

In Africa and many other places, the impact caused by animal diseases continues to negatively affect the local economy. Dermatophilosis is a tick-borne disease of ruminants and other animals [5] and affects all parts of the body of the animal. In Nigeria and Cameroon, Dermatophilosis accounts for about 75% of morbidity in herds and about 12% in cattle. Mortality rate has been reported to be quite high due to the resulting toxemia and general debility [5]. Dermatophilosis is an intractable disease and highly contagious, spreading from cattle to man (zoonotic).

The common and orthodox treatment for dermatophilosis is through the use of classical antibiotics like lamstreptocide, charmil, and terramycin long acting (TLA), 1% potassium aluminum sulphate dip, and co-biotic (penicillin and dihydrostreptomycin). Apart from the toxicity of some of these drugs, some of them contain heavy metals, which on accumulation could cause tumors and cancers in both man and animals [3, 6]. The use of organophosphate dips has also been reported to have a negative effect on the environment, and it has been observed to cause systemic damages on internal organs of both animals and humans.

**23**

**Figure 1.**

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa*

**3. Plant pathogenic fungal contamination of food and crops in Africa**

of field crops and harvested products. Most of these plant pathogenic diseases are fungi. Despite the availability of chemicals for control of these pathogens, many farmers find it inaccessible for reasons of costs and lack of adequate knowledge on usage. In Africa, the predominant food source for more than 70% of the population is grains such as maze and groundnuts. Even though there are new and improved methods for containing these diseases in food crops, there are still great losses due to fungal infections of the crops. A number of reports have shown that aflatoxins producing fungi are predominant with both field and stored maize and groundnuts. Aflatoxin (Aflatoxin B1) is produced by *Aspergillus flavus* or *Aspergillus parasiticus*, and effects of aflatoxin on crops like maize and groundnuts completely destroy the

Agricultural plant products in sub-Saharan Africa often decay fast due to infection

Traditional cultivation techniques were employed for isolation and identification. Swab samples were taken from the lesions on the animal and analyzed at the Phytobiotechnology Research laboratory for *Dermatophilus congolensis* culture. Initial cultures were done in thioglycolate broth and subculture after 48 hours on fortified chloramphenicol Sabouraud Dextrose agar and modified cycloheximide (Actidione)-chloramphenicol Sabouraud agar previously prepared and incubated for 48 hours at 35°C in an aerobic condition. For specific cultural distinct features on agar plate, blood and chocolate agar plates were prepared and distinct colonies from Sabouraud agar plates were transferred on to blood and chocolate agar plates aseptically incubated in air supplemented with 5% CO2, and the blood

At 24 hours, a pure culture with tiny, point-like, smooth, creamy, white-colored, beta-hemolytic colonies adherent to the media grew in aerobic blood agar and chocolate agar, with Gram staining showing hypha-like, branching filaments with "train track" forms and clusters of sporangia as well as Gram-positive coccoid forms, mostly in chains. After 48 h, crowded colonies became yellowish and mucoid, with a great variation in colonial morphology, for example, pulvinate, umbonate, or cake crumb-like forms were considered typical of *Dermatophilus congolensis*. This is

*Beta-hemolytic colonies after 2 days of incubation at 37°C on blood agar medium, with pleomorphic appearance in pulvinate, umbonate, or cake crumb-like form. Dermatophilus congolensis on blood agar plate.*

*DOI: http://dx.doi.org/10.5772/intechopen.81692*

crop due to the toxicity of aflatoxin to humans.

**4.1 Isolation and identification of** *Dermatophilus congolensis*

agar was also incubated in an anaerobic atmosphere [4, 7].

**4. Methodology**

shown in **Figures 1** and **2**.

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa DOI: http://dx.doi.org/10.5772/intechopen.81692*

#### **3. Plant pathogenic fungal contamination of food and crops in Africa**

Agricultural plant products in sub-Saharan Africa often decay fast due to infection of field crops and harvested products. Most of these plant pathogenic diseases are fungi. Despite the availability of chemicals for control of these pathogens, many farmers find it inaccessible for reasons of costs and lack of adequate knowledge on usage.

In Africa, the predominant food source for more than 70% of the population is grains such as maze and groundnuts. Even though there are new and improved methods for containing these diseases in food crops, there are still great losses due to fungal infections of the crops. A number of reports have shown that aflatoxins producing fungi are predominant with both field and stored maize and groundnuts. Aflatoxin (Aflatoxin B1) is produced by *Aspergillus flavus* or *Aspergillus parasiticus*, and effects of aflatoxin on crops like maize and groundnuts completely destroy the crop due to the toxicity of aflatoxin to humans.

#### **4. Methodology**

*Biotechnology and Bioengineering*

ably organic, is needed [3].

**2. Dermatophilosis in animals**

occasionally humans [5].

are very common across the world, particularly in wet areas like Cameroon. The ecology of Cameroon is rich in algae and lichens; bryophytes in Cameroon are part of the Congo forest and the highlands from Mount Cameroon via the Atlantika Mountains to the Mandara Mountains collectively constituting the Congo forest from Nigeria, ranges from 1400 to 4000 m, and harbors a rich biodiversity of both lower and higher plants. A survey of bryophytes in Cameroon revealed many unidentified species with familiar dormant species such as *Marchantia spp* [2].

Phenanthrenes and other phenolics have been isolated from in vitro cultures of *Marchantia polymorpha.* Recently, extensive report was published on the biology and constituents and chemistry and organic natural products of bryophytes [1, 4], though this lacks data on bryophytes from West and Central Africa, especially Cameroon. Screening of bryophytes and lower plants for biologically active compounds from Cameroon is of great importance considering that Cameroon is centrally placed in Africa and harbors all the ecological and geographical characteristics widespread across Africa. The search for bioactive compounds from plants in the past 30 years in Africa has concentrated on higher plants with little or no interest on bryophytes, which is the same in the rest of the world [2]. The prevalence of many plant and animal pathogenic diseases is growing along with drug resistance strains. This generates huge losses in agricultural yield and productivity across Africa. Treatment and management are expensive for many African farmers and therefore a cheap alternative, prefer-

Bioactive compounds from bryophytes could bridge this gap. Here, we show that new drug leads could be identified from bryophytes from Cameroon to address plant pathogenic diseases and animal diseases like *Dermatophilosis* infection in cattle.

Dermatophilosis is caused by the bacterium *Dermatophilus congolensis*, which is an aerobic actinomycete (facultatively anaerobic) and usually affects animals and

Dermatophilosis is distributed worldwide, prevailing in tropical areas, and related to humid environments and other factors, such as poor veterinary services, coinfection with a number of bacterial infections, especially in animals with compromised immune systems, and poor hygiene conditions in favor of its occurrence and spread. In Africa and many other places, the impact caused by animal diseases continues to negatively affect the local economy. Dermatophilosis is a tick-borne disease of ruminants and other animals [5] and affects all parts of the body of the animal. In Nigeria and Cameroon, Dermatophilosis accounts for about 75% of morbidity in herds and about 12% in cattle. Mortality rate has been reported to be quite high due to the resulting toxemia and general debility [5]. Dermatophilosis is an intractable

The common and orthodox treatment for dermatophilosis is through the use of classical antibiotics like lamstreptocide, charmil, and terramycin long acting (TLA), 1% potassium aluminum sulphate dip, and co-biotic (penicillin and dihydrostreptomycin). Apart from the toxicity of some of these drugs, some of them contain heavy metals, which on accumulation could cause tumors and cancers in both man and animals [3, 6]. The use of organophosphate dips has also been reported to have a negative effect on the environment, and it has been observed to cause systemic damages on internal organs of both animals and

disease and highly contagious, spreading from cattle to man (zoonotic).

**22**

humans.

#### **4.1 Isolation and identification of** *Dermatophilus congolensis*

Traditional cultivation techniques were employed for isolation and identification. Swab samples were taken from the lesions on the animal and analyzed at the Phytobiotechnology Research laboratory for *Dermatophilus congolensis* culture. Initial cultures were done in thioglycolate broth and subculture after 48 hours on fortified chloramphenicol Sabouraud Dextrose agar and modified cycloheximide (Actidione)-chloramphenicol Sabouraud agar previously prepared and incubated for 48 hours at 35°C in an aerobic condition. For specific cultural distinct features on agar plate, blood and chocolate agar plates were prepared and distinct colonies from Sabouraud agar plates were transferred on to blood and chocolate agar plates aseptically incubated in air supplemented with 5% CO2, and the blood agar was also incubated in an anaerobic atmosphere [4, 7].

At 24 hours, a pure culture with tiny, point-like, smooth, creamy, white-colored, beta-hemolytic colonies adherent to the media grew in aerobic blood agar and chocolate agar, with Gram staining showing hypha-like, branching filaments with "train track" forms and clusters of sporangia as well as Gram-positive coccoid forms, mostly in chains. After 48 h, crowded colonies became yellowish and mucoid, with a great variation in colonial morphology, for example, pulvinate, umbonate, or cake crumb-like forms were considered typical of *Dermatophilus congolensis*. This is shown in **Figures 1** and **2**.

#### **Figure 1.**

*Beta-hemolytic colonies after 2 days of incubation at 37°C on blood agar medium, with pleomorphic appearance in pulvinate, umbonate, or cake crumb-like form. Dermatophilus congolensis on blood agar plate.*

#### **Figure 2.**

*Gram stain with characteristic branching filaments with "train track" forms or hypha-like chains that released sporangium Gram-positive cells (magnification, 1000×). Beta-hemolytic colonies after 2 days of incubation at 37°C on blood agar medium, with pleomorphic appearance in pulvinate, umbonate, or cake crumb-like form.*

**Figures 1** and **2** reveal the unique, distinct bacteriological features of *Dermatophilus congolensis.* The biochemical characteristics of *D congolensis* as basis for identification done according Monica Cheesbrough [8] revealed that beta hemolysis in 3–7 days, oxidase, gelatin, casein and starch all positive, while *D congolensis* fermented fructose, ribose and galactose.

#### **5. Survey and extraction of bryophytes**

A preliminary survey of liverworts in northwest and southwest regions of Cameroon was performed. Bryophytes (species of liverwort and moss) from Cameroon West/Central Africa were collected and complete sequences for the 18S-rRNA gene of bryophytes were used to construct a phylogenetic tree of bryophytes from Cameroon to fully identify the prevalent species in Cameroon.

#### **5.1 Extraction procedures for the selected and identified bryophyte species**

About 50 g of each of the bryophyte (*Marchantia debilis* and Plangiochila spp) plant material were added separately to 250 ml each of methanol and petroleum ether (1:5 w/v) in 250 beakers (Pyrex) for each plant mash and allowed to extract for 72 hours [6]. The extracts were filtered by gravity filtration using Whatman filter paper no 1 locally purchased in Bamenda, Cameroon, and the filtrate solvent was evaporated under vacuum using an incubator at 37°C and the resulting dried extracts were stored in sterile screw-capped bottles and kept at room temperature for further antibacterial testing using extracts of bryophytes. The morphology of the bryophytes and the nature of extracts is shown in **Figures 3**–**8**.

#### **5.2 Antibacterial activity of the extracts of bryophytes**

The agar diffusion method according to Yongabi et al. [9] was employed. Around 0.2 g of the *Marchantia debilis* and *Plangiochila* spp. extracts was reconstituted in 5 ml of distilled water. Antibiotic susceptibility will be determined by agar well diffusion method—commonly used and standardized in the US by National Committee for Clinical Laboratory Standards (NCCLS) [8, 10].

The zone of inhibition was measured and results interpreted as sensitive, intermediate resistant, or resistant. The zone sizes of inhibition were measured and interpreted using the NCCLS as recommended by WHO [8]. Each of the extracts was incorporated in a 6-mm well previously bored using a steel borer. A control set up was established by introducing the extracting solvent (methanol and petroleum) into the

**25**

**Table 2.**

*microbes.*

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa*

different wells as well. The plates were incubated at 37°C for 36 hours. The development of inhibition by the extracts against the test organism was measured [11].

**5.3 Preparation of bryophyte extracts-based ointment using olive oil base**

The organic extracts (200 mg each) of *Marchantia* spp. and *Plangiochila* were blended into 200 ml of olive oil and palm kernel oil. Standard organic chemistry

**Fresh** *Marchantia debilis* **Dried** *Marchantia debilis*

9 mm 12 mm 14 mm 9.5 mm 17.2 mm 12.5 mm

0 mm 0 mm 0 mm 10.5 mm 5.2 mm 12.5 mm

5 mm 5 mm 13 mm 6 mm 11 mm 12.5 mm

**Fresh** *Plangiochila spp* **Dried** *Plangiochila spp*

0 mm 0 mm 0 mm 0 mm 0 mm 8.9 mm

6 mm 6 mm 7 mm 7 mm 8 mm 9.5 mm

**Hexane extracts**

**Hexane extracts** **Petroleum extracts**

2 cm 1 cm No

**Petroleum extracts**

0.5 cm 0.5 cm No Growth

**Methanol extracts**

Growth

**Methanol extracts**

**Methanol extracts**

*Bacillus spp* 0 mm 0 mm 0 mm 11.5 mm 6.2 mm 13.5 mm

Growth

**Methanol extracts**

*Staphylococcus aureus* 9 mm 8 mm 14 mm 9.8 mm 8.8 mm 15.5 mm

*Bacillus spp* 0 mm 0 mm 0 mm 0 mm 0 mm 5 mm

Growth

*Dermatophilus congolensis is an isolate from cow. For Aspergillus flavus (an isolate from maize rot) the inhibition is* 

*Preliminary in vitro test showing average zone of inhibition of organic extracts of Plangiochila spp. on different* 

*Dermatophilus congolensis is an isolate from cow. For Aspergillus flavus (an isolate from maize rot) the inhibition is* 

*Preliminary in vitro test showing zone of inhibition of organic extracts of Marchantia debilis on different* 

**Petroleum extracts**

The differences between the inhibition rates of the extracts in the test setup and that of the control were recorded as actual diameter of zones of inhibition caused by the extract. The methanol and petroleum extracts did not exhibit any inhibition

*DOI: http://dx.doi.org/10.5772/intechopen.81692*

in this study, as shown in **Tables 1** and **2**.

**Hexane extracts**

*Aspergillus flavus* 3 cm 2 cm No

*given as growth of fungi in 7 days, where the control grew 10 cm.*

**Hexane extracts**

*Aspergillus flavus* 1 cm 1 cm No

*given as growth of fungi in 7 days, where the control grew 10 cm.*

**Microbial test organism**

*Staphylococcus aureus*

*Pseudomonas aeruginosa*

*Dermatophilus congolensis*

**Table 1.**

*microbes.*

**Microbial test organism**

*Pseudomonas aeruginosa*

*Dermatophilus congolensis*

protocols as described by Yongabi et al. [9] were applied.

**Petroleum extracts**

#### *Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa DOI: http://dx.doi.org/10.5772/intechopen.81692*

different wells as well. The plates were incubated at 37°C for 36 hours. The development of inhibition by the extracts against the test organism was measured [11].

The differences between the inhibition rates of the extracts in the test setup and that of the control were recorded as actual diameter of zones of inhibition caused by the extract. The methanol and petroleum extracts did not exhibit any inhibition in this study, as shown in **Tables 1** and **2**.

#### **5.3 Preparation of bryophyte extracts-based ointment using olive oil base**

The organic extracts (200 mg each) of *Marchantia* spp. and *Plangiochila* were blended into 200 ml of olive oil and palm kernel oil. Standard organic chemistry protocols as described by Yongabi et al. [9] were applied.


*Dermatophilus congolensis is an isolate from cow. For Aspergillus flavus (an isolate from maize rot) the inhibition is given as growth of fungi in 7 days, where the control grew 10 cm.*

#### **Table 1.**

*Biotechnology and Bioengineering*

**Figure 2.**

**Figures 1** and **2** reveal the unique, distinct bacteriological features of *Dermatophilus congolensis.* The biochemical characteristics of *D congolensis* as basis for identification done according Monica Cheesbrough [8] revealed that beta hemo-

*Gram stain with characteristic branching filaments with "train track" forms or hypha-like chains that released sporangium Gram-positive cells (magnification, 1000×). Beta-hemolytic colonies after 2 days of incubation at 37°C on blood agar medium, with pleomorphic appearance in pulvinate, umbonate, or cake crumb-like form.*

A preliminary survey of liverworts in northwest and southwest regions of Cameroon was performed. Bryophytes (species of liverwort and moss) from Cameroon West/Central Africa were collected and complete sequences for the 18S-rRNA gene of bryophytes were used to construct a phylogenetic tree of bryophytes from Cameroon to fully identify the prevalent species in Cameroon.

**5.1 Extraction procedures for the selected and identified bryophyte species**

The agar diffusion method according to Yongabi et al. [9] was employed. Around 0.2 g of the *Marchantia debilis* and *Plangiochila* spp. extracts was reconstituted in 5 ml of distilled water. Antibiotic susceptibility will be determined by agar well diffusion method—commonly used and standardized in the US by National

The zone of inhibition was measured and results interpreted as sensitive, intermediate resistant, or resistant. The zone sizes of inhibition were measured and interpreted using the NCCLS as recommended by WHO [8]. Each of the extracts was incorporated in a 6-mm well previously bored using a steel borer. A control set up was established by introducing the extracting solvent (methanol and petroleum) into the

the bryophytes and the nature of extracts is shown in **Figures 3**–**8**.

Committee for Clinical Laboratory Standards (NCCLS) [8, 10].

**5.2 Antibacterial activity of the extracts of bryophytes**

About 50 g of each of the bryophyte (*Marchantia debilis* and Plangiochila spp) plant material were added separately to 250 ml each of methanol and petroleum ether (1:5 w/v) in 250 beakers (Pyrex) for each plant mash and allowed to extract for 72 hours [6]. The extracts were filtered by gravity filtration using Whatman filter paper no 1 locally purchased in Bamenda, Cameroon, and the filtrate solvent was evaporated under vacuum using an incubator at 37°C and the resulting dried extracts were stored in sterile screw-capped bottles and kept at room temperature for further antibacterial testing using extracts of bryophytes. The morphology of

lysis in 3–7 days, oxidase, gelatin, casein and starch all positive, while

*D congolensis* fermented fructose, ribose and galactose.

**5. Survey and extraction of bryophytes**

**24**

*Preliminary in vitro test showing zone of inhibition of organic extracts of Marchantia debilis on different microbes.*


*Dermatophilus congolensis is an isolate from cow. For Aspergillus flavus (an isolate from maize rot) the inhibition is given as growth of fungi in 7 days, where the control grew 10 cm.*

#### **Table 2.**

*Preliminary in vitro test showing average zone of inhibition of organic extracts of Plangiochila spp. on different microbes.*

**Figure 3.** *Marchantia debilis (Liverwort).*

**Figure 4.** *Plangiochila spp (Moss).*

**Figure 5.**

*Two samples of fresh Marchantia debilis residues after extraction with hexane and petroleum ether.*

#### **Figure 6.**

*Two samples of partially dried Plangiochila spp residues after extraction with hexane and petroleum ether.*

**27**

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa*

**5.4 Ointment application and resultant outcome of application**

*Partially dried Marchantia debilis extract in hexane and in petroleum ether.*

dead skin was then carefully peeled off.

*Extract of Plangiochila spp in hexane and petroleum ether.*

*debilis* from Cameroon [2] (**Figures 10** and **11**).

**of this study**

**Figure 7.**

**Figure 8.**

An animal health officer applied the cream topically (by rubbing on affected parts of the animal, using hand gloves) once a day for 3 days in a week. Following this, a total drying off of the infected spot was noticed after 14 days. The dried,

**6. The findings, discussion on the economic and environmental benefits** 

The results show that extracts of *Marchantia debilis* and *Plangiochila* have antifungal activity against *Aspergillus flavus*, and antibacterial activities against *Pseudomonas* spp., *Bacillus spp*, and *Staphylococcus aureus* and *Dermatophilus congolensis* isolates (**Tables 1** and **2**). In **Figure 9**, a plate that demonstrates clear mycelia inhibition of *Aspergillus flavus* by extract of *Marchantia debilis* is shown. The product development focus has been on *Marchantia debilis* since Yongabi et al. [2] isolated a number of marchantins including a new marchantin Q from *Marchantia* 

Antimicrobial activity of liverworts is not new [1] but the testing of these liverworts and moss on isolates from plant pathogens in Africa is probably for the first time. The *Aspergillus flavus* isolate was provided for this study by a local laboratory in Cameroon. The effect of liverworts inhibiting the growth of *Dermatophilus congolensis* isolated from cattle is reported here for the first time. The synthetic agrochemicals used in daily farming in Africa are quite expensive and these synthetic products are normally out of reach of the rural farmer [5, 9, 12–14]. The result

*DOI: http://dx.doi.org/10.5772/intechopen.81692*

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa DOI: http://dx.doi.org/10.5772/intechopen.81692*

#### **Figure 7.**

*Biotechnology and Bioengineering*

**Figure 3.**

**Figure 4.**

**Figure 5.**

*Two samples of fresh Marchantia debilis residues after extraction with hexane and petroleum ether.*

*Two samples of partially dried Plangiochila spp residues after extraction with hexane and petroleum ether.*

*Plangiochila spp (Moss).*

*Marchantia debilis (Liverwort).*

**26**

**Figure 6.**

*Partially dried Marchantia debilis extract in hexane and in petroleum ether.*

**Figure 8.** *Extract of Plangiochila spp in hexane and petroleum ether.*

#### **5.4 Ointment application and resultant outcome of application**

An animal health officer applied the cream topically (by rubbing on affected parts of the animal, using hand gloves) once a day for 3 days in a week. Following this, a total drying off of the infected spot was noticed after 14 days. The dried, dead skin was then carefully peeled off.

#### **6. The findings, discussion on the economic and environmental benefits of this study**

The results show that extracts of *Marchantia debilis* and *Plangiochila* have antifungal activity against *Aspergillus flavus*, and antibacterial activities against *Pseudomonas* spp., *Bacillus spp*, and *Staphylococcus aureus* and *Dermatophilus congolensis* isolates (**Tables 1** and **2**). In **Figure 9**, a plate that demonstrates clear mycelia inhibition of *Aspergillus flavus* by extract of *Marchantia debilis* is shown. The product development focus has been on *Marchantia debilis* since Yongabi et al. [2] isolated a number of marchantins including a new marchantin Q from *Marchantia debilis* from Cameroon [2] (**Figures 10** and **11**).

Antimicrobial activity of liverworts is not new [1] but the testing of these liverworts and moss on isolates from plant pathogens in Africa is probably for the first time. The *Aspergillus flavus* isolate was provided for this study by a local laboratory in Cameroon. The effect of liverworts inhibiting the growth of *Dermatophilus congolensis* isolated from cattle is reported here for the first time. The synthetic agrochemicals used in daily farming in Africa are quite expensive and these synthetic products are normally out of reach of the rural farmer [5, 9, 12–14]. The result

#### **Figure 9.**

*Plate labeled marked (MAR) contains extract of Marchantia debilis Goebel with slow growth of Aspergillus flavus in 7 days as opposed to control plate (Marked: Control) with only n-hexane incorporated into the agar.*

#### **Figure 10.**

*A cured cow: management of Dermatophilosis in ruminants using Bryo-ointment.*

**29**

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa*

is that a lot of agricultural produce such as cattle and grains are lost to disease and the cost of these produce is prohibitive. More so because grains are not stored for a

The problem in cattle with Dermatophilosis is no different. In a lot of the developing countries, today, the problem of malnutrition is endemic and the related opportunistic infections lead to infectious disease, such as tuberculosis and malaria. Protein malnutrition in Africa is a serious problem, especially in rural Africa where approximately 70% of the population live [4, 7, 18, 19]. The chemical constituents of bryophytes are well studied [1, 2], but these rich chemical constituents have not yet been explored biotechnologically. Plants with bioactive ingredients abound in

The production of plant-based products from the bryophytes in the treatment of Dermatophilosis shifts focus from the importation of orthodox drugs and conserves Africa's scarce foreign exchange reserve, and increases utilization of indigenous plant resources. Outside of the cheaper ointment product, a local industry for the production of this ointment is encouraged and the product would be available to a larger group of herders. This preliminary report details the first attempt. The multiplier effect is enormous; meat should be cheaper and malnutrition resulting from the lack of protein would drastically reduce, and possibly disappear. Moreover, the use of plant-based products could easily foreclose the emergent, resistant strains of *Dermatophilus congolensis* resulting from the frequent

Bryophytes are common and abundant, especially in the west and central African regions. The formulation process for the ointment is easy to follow and it is based on a technology that the rural populations could easily handle. The method of application is by glove-protected hands to animals, and the ointment is also effec-

to have astringent property when applied on sores. Thus, it not only heals but also smoothens the affected lesion to which it is applied. The oils from bryophytes when blended with *Vitellaria paradoxa* have improved cosmetic value and reduced treatment time, and this gives the formulated cream an added advantage. The Bryo-ointment is cheap and effective. For instance, 100 ml is sold at 1 US dollar, as against 5 US dollars for each of the antibiotics. Based on our findings, a container of 100 ml of the formulated cream can hardly be used completely in treating three animals once a day for 3 days a week, and for 2 weeks. However, this depends on the degree of infection.

The ointment and bryosol (bryophyte solution suspended in glycerol) are observed

The findings of the study suggest that one cannot begin addressing the problem of aflatoxin producing fungi on crops, grains contamination, and skin diseases in animals by simply relying on agrochemicals and introducing improved management practices. This requires a closer examination of the role of ecological technologies and approaches. Above all, studies on bryophytes are limited to taxonomy and molecular biological aspects with little effort toward actual biotransformation of bryophytes via appropriate biotechnology for direct applications in horticulture

• A shift toward cost-effective technology will not take place unless a series of interventions via technology such as bryo biotechnology that can give neces-

sary opportunities is provided to the farmers and other stakeholders.

*DOI: http://dx.doi.org/10.5772/intechopen.81692*

use and misuse of antibiotics.

**7. Recommendations**

and animal husbandry.

tive against human skin infections [9, 14, 21, 22].

From this study, it is therefore recommend that:

longer period to enable sales at off season [15–17].

Africa [20], and bryophytes are even more abundant [3].

#### *Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa DOI: http://dx.doi.org/10.5772/intechopen.81692*

is that a lot of agricultural produce such as cattle and grains are lost to disease and the cost of these produce is prohibitive. More so because grains are not stored for a longer period to enable sales at off season [15–17].

The problem in cattle with Dermatophilosis is no different. In a lot of the developing countries, today, the problem of malnutrition is endemic and the related opportunistic infections lead to infectious disease, such as tuberculosis and malaria. Protein malnutrition in Africa is a serious problem, especially in rural Africa where approximately 70% of the population live [4, 7, 18, 19]. The chemical constituents of bryophytes are well studied [1, 2], but these rich chemical constituents have not yet been explored biotechnologically. Plants with bioactive ingredients abound in Africa [20], and bryophytes are even more abundant [3].

The production of plant-based products from the bryophytes in the treatment of Dermatophilosis shifts focus from the importation of orthodox drugs and conserves Africa's scarce foreign exchange reserve, and increases utilization of indigenous plant resources. Outside of the cheaper ointment product, a local industry for the production of this ointment is encouraged and the product would be available to a larger group of herders. This preliminary report details the first attempt. The multiplier effect is enormous; meat should be cheaper and malnutrition resulting from the lack of protein would drastically reduce, and possibly disappear. Moreover, the use of plant-based products could easily foreclose the emergent, resistant strains of *Dermatophilus congolensis* resulting from the frequent use and misuse of antibiotics.

Bryophytes are common and abundant, especially in the west and central African regions. The formulation process for the ointment is easy to follow and it is based on a technology that the rural populations could easily handle. The method of application is by glove-protected hands to animals, and the ointment is also effective against human skin infections [9, 14, 21, 22].

The ointment and bryosol (bryophyte solution suspended in glycerol) are observed to have astringent property when applied on sores. Thus, it not only heals but also smoothens the affected lesion to which it is applied. The oils from bryophytes when blended with *Vitellaria paradoxa* have improved cosmetic value and reduced treatment time, and this gives the formulated cream an added advantage. The Bryo-ointment is cheap and effective. For instance, 100 ml is sold at 1 US dollar, as against 5 US dollars for each of the antibiotics. Based on our findings, a container of 100 ml of the formulated cream can hardly be used completely in treating three animals once a day for 3 days a week, and for 2 weeks. However, this depends on the degree of infection.

#### **7. Recommendations**

The findings of the study suggest that one cannot begin addressing the problem of aflatoxin producing fungi on crops, grains contamination, and skin diseases in animals by simply relying on agrochemicals and introducing improved management practices. This requires a closer examination of the role of ecological technologies and approaches. Above all, studies on bryophytes are limited to taxonomy and molecular biological aspects with little effort toward actual biotransformation of bryophytes via appropriate biotechnology for direct applications in horticulture and animal husbandry.

From this study, it is therefore recommend that:

• A shift toward cost-effective technology will not take place unless a series of interventions via technology such as bryo biotechnology that can give necessary opportunities is provided to the farmers and other stakeholders.

*Biotechnology and Bioengineering*

*Plate labeled marked (MAR) contains extract of Marchantia debilis Goebel with slow growth of Aspergillus flavus in 7 days as opposed to control plate (Marked: Control) with only n-hexane incorporated into the agar.*

*A cured cow: management of Dermatophilosis in ruminants using Bryo-ointment.*

*Maize drying sprayed with Bryo-extracts to inhibit moldy cotton wool Aspergillus flavus.*

**Figure 9.**

**Figure 10.**

**28**

**Figure 11.**


#### **Glossary**


### **Author details**

Kenneth Yongabi Anchang1,2,3,4\* and Henrik Toft Simonsen5

1 Public Health Infectiology and Phytobiotechnology, Imo State University, Owerri, Nigeria


\*Address all correspondence to: yongabika@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**31**

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa*

*Mycobacterium avium* complex isolated from AIDS patients by Garlic. Journal of Antimicrobial Chemotherapy.

fungicidal and fungistatic effects of an aqueous garlic extracts on medically important yeasts and fungi. Mycologia.

[11] Moore GS, Atkins RD. The

[12] Arthur GH, Alen WR. Swine bacterial infections. Equine Veterinary

[13] Arthur GH, Noakes D, Pearson H. Veterinary Reproductive and Obstetrics. 5th ed. London: Bailliere Tyndall; 1982.

[14] Yongabi KA, Agho MO, Chindo IY, Dukku UH. Studies on the

antifungal properties of *Urtica dioica*; *Urticaceeae* (stinging nettle). Journal of Phytomedicine and Therapeutics.

SM. Dermatophilosis in Northern Nigeria. The Veterinary Bulletin.

KC. Dermatophilosis infection in animals and man. In: Edited Papers from 1976, Ibadan Conference. 1976

[17] Railey J, George Mandel H, Sinha S, Judahand DL, Neal GE. Invitro activation of human Harvey-ras Proto Onco gene by aflatoxin b1. Carcinogensis. 1997;**18**:905-910

[18] Dave EL. Chapter 9.26: Aflatoxin toxicology. In: Comprehensive

Toxicology. UK: Pergamon Publications;

[19] Groopmann JD, Kensler TW**.** CRC Critical Reviews in Toxicology. Chapter

19. Ghana: 1999. pp. 113-124

Journal. 1972;**4**(109):15-17

1993;**32**:623-626

1977;**69**:341-348

pp. 501-509

2000;**5**(1):39-43

1972;**46**:471-478

1997

[15] Bida SA, Dennis

[16] Lloyd DH, Sellers

*DOI: http://dx.doi.org/10.5772/intechopen.81692*

[1] Asakawa Y. Chemical Constituents of Bryophytes Progress in the Chemistry of Organic Natural Products. Vol. 95. Singapore: Springer-Verlag wien; 2013. DOI: 10.1007/978-3-7091-1084-3

[2] Yongabi KY, Novakovic M, Bukvicki D, Asakawa Y. Bis-bibenzyls from the Cameroon Liverwort *Marchantia debilis*. Natural Product Communications.

[3] Am A, Paul C, Konig WA, Muhle H. Volatile constituents in the liverwort *Tritomaria polita*. Phytochemistry.

[4] Adam K-P, Becker H. Phenanthrenes and other Phenolics from in vitro cultures of *Marchantia polymorphs*. Phytochemistry. 1994;**35**:139

[5] Abdulkadir IA. Infectious Diseases of Livestock in Nigeria. An Outline. Vol. 274-279. Nigeria: ABU Press Limited;

[6] Irobi ON, Daramola SO. Antifungal activities of crude extract of *Mitracarpus villosus*. Journal of Ethnopharmacology.

[7] Allison KW, Chud J. The Liverworts of a New Zealand. Dunedin: University

[8] Cheesbrough M. Medical laboratory

[9] Yongabi KA, Agho MO, Chindo Y, Buba MW. Evaluating the medicinal potentials of some indigenous plants in controlling microbial

contamination of poultry feed. Journal of Phytomedicine and Therapeutics.

[10] Deshpande RG, Khan MB, Bhat DA, Navalkar RG. Inhibition of

of Otugo Press; 1975. p. 300

manual for tropical countries. Microbiology, Butterworth's.

**References**

2016;**11**(9):1317-1318

2003;**64**:637

1989. pp. 37-40

1993;**40**:137-140

1984;**2**:76-135

2000;**5**(2):98-102

*Developments and Perspectives in Bryophyte Biotechnology in Sub-Saharan Africa DOI: http://dx.doi.org/10.5772/intechopen.81692*

#### **References**

*Biotechnology and Bioengineering*

**30**

**Author details**

**Glossary**

Nigeria

provided the original work is properly cited.

2 Ebonyi State University, Abakaliki, Nigeria

Kenneth Yongabi Anchang1,2,3,4\* and Henrik Toft Simonsen5

4 Catholic University of Cameroon, Bamenda, Cameroon

\*Address all correspondence to: yongabika@yahoo.com

5 DTU Bioengineering, Technical University of Denmark, Denmark

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Public Health Infectiology and Phytobiotechnology, Imo State University, Owerri,

• Bryophyte bioproducts offer an opportunity for sustainable animal husbandry

• Though farmers pay considerable attention to the selection of seed from their own produce, lack of awareness about identification of contamination in general prevents them from using aflatoxin-free seeds. Interventions such as treating grains with bryophyte-derived solution may ensure that farmers use

grains free from contamination irrespective of the sources of supply.

Dermatophilosis bacterial skin disease of cows, sheep, goats, dogs and other animals, scab disease

*Amblyomma variegatum* tick vector of Dermatophilosis (cutaneous streptothricosis) *Dermatophilus congolensis* the bacterial causative agent of Dermatophilosis

Lamstreptocide antibiotics used in treatment of Dermatophilosis TLA terramycin long-acting antibiotic used in the treatment of Dermatophilosis Zoonotic animal disease that could also be passed to infect man Necrosis the breaking down of cells/tissues resulting from an

Ecology the study of the relations of living things to one

another and to their surrounding

Kirchi Hausa name for Dermatophilosis

infection Hausa the language of Hausa people in Nigeria and Cameroon

and agriculture for Africans at potentially lower costs.

3 Phyto-biotechnology Research Foundation Clinic/Institute, Bamenda, Cameroon

[1] Asakawa Y. Chemical Constituents of Bryophytes Progress in the Chemistry of Organic Natural Products. Vol. 95. Singapore: Springer-Verlag wien; 2013. DOI: 10.1007/978-3-7091-1084-3

[2] Yongabi KY, Novakovic M, Bukvicki D, Asakawa Y. Bis-bibenzyls from the Cameroon Liverwort *Marchantia debilis*. Natural Product Communications. 2016;**11**(9):1317-1318

[3] Am A, Paul C, Konig WA, Muhle H. Volatile constituents in the liverwort *Tritomaria polita*. Phytochemistry. 2003;**64**:637

[4] Adam K-P, Becker H. Phenanthrenes and other Phenolics from in vitro cultures of *Marchantia polymorphs*. Phytochemistry. 1994;**35**:139

[5] Abdulkadir IA. Infectious Diseases of Livestock in Nigeria. An Outline. Vol. 274-279. Nigeria: ABU Press Limited; 1989. pp. 37-40

[6] Irobi ON, Daramola SO. Antifungal activities of crude extract of *Mitracarpus villosus*. Journal of Ethnopharmacology. 1993;**40**:137-140

[7] Allison KW, Chud J. The Liverworts of a New Zealand. Dunedin: University of Otugo Press; 1975. p. 300

[8] Cheesbrough M. Medical laboratory manual for tropical countries. Microbiology, Butterworth's. 1984;**2**:76-135

[9] Yongabi KA, Agho MO, Chindo Y, Buba MW. Evaluating the medicinal potentials of some indigenous plants in controlling microbial contamination of poultry feed. Journal of Phytomedicine and Therapeutics. 2000;**5**(2):98-102

[10] Deshpande RG, Khan MB, Bhat DA, Navalkar RG. Inhibition of

*Mycobacterium avium* complex isolated from AIDS patients by Garlic. Journal of Antimicrobial Chemotherapy. 1993;**32**:623-626

[11] Moore GS, Atkins RD. The fungicidal and fungistatic effects of an aqueous garlic extracts on medically important yeasts and fungi. Mycologia. 1977;**69**:341-348

[12] Arthur GH, Alen WR. Swine bacterial infections. Equine Veterinary Journal. 1972;**4**(109):15-17

[13] Arthur GH, Noakes D, Pearson H. Veterinary Reproductive and Obstetrics. 5th ed. London: Bailliere Tyndall; 1982. pp. 501-509

[14] Yongabi KA, Agho MO, Chindo IY, Dukku UH. Studies on the antifungal properties of *Urtica dioica*; *Urticaceeae* (stinging nettle). Journal of Phytomedicine and Therapeutics. 2000;**5**(1):39-43

[15] Bida SA, Dennis SM. Dermatophilosis in Northern Nigeria. The Veterinary Bulletin. 1972;**46**:471-478

[16] Lloyd DH, Sellers KC. Dermatophilosis infection in animals and man. In: Edited Papers from 1976, Ibadan Conference. 1976

[17] Railey J, George Mandel H, Sinha S, Judahand DL, Neal GE. Invitro activation of human Harvey-ras Proto Onco gene by aflatoxin b1. Carcinogensis. 1997;**18**:905-910

[18] Dave EL. Chapter 9.26: Aflatoxin toxicology. In: Comprehensive Toxicology. UK: Pergamon Publications; 1997

[19] Groopmann JD, Kensler TW**.** CRC Critical Reviews in Toxicology. Chapter 19. Ghana: 1999. pp. 113-124

[20] Sofowora A. Medicinal Plants and Traditional in Africa. Part 11. Ife, Nigeria: Pitman Pess Ltd; 1984. p. 128, 142, 146

[21] Njoku CO, Alafiatayo RA. Comparative pathology of the main bovine skin disease in Nigeria. In: A paper presented at the National Conference on Disease of Ruminants, NVRI, Vom. 1984

[22] Ames BN, Profet M, Gold LS. Nature's chemicals and synthetic chemicals. Comparative Toxicology PNAS. 1990;**87**:7782-7786

**33**

**Chapter 4**

**Abstract**

prokaryotes

**1. Introduction**

Hypotoxic Fluorescent

Mammalians Cells

Nanoparticles Delivery by Cell-

Penetrating Peptides in Multiple

Organisms: From Prokaryotes to

Nanotechnology is the study of materials in the nanoscale. By its nature, nanotechnology is interdisciplinary. Nanotechnology has made a significant stride in recent two decades in various industries. Numerous nanomaterials are devised for biomedical applications which include intracellular tracking and labeling, gene detection and hybridization, tumor or tissue targeting, pharmaceutical therapies, pathogenic inhibiting, and medical instrument coating for disinfections. High photostability and quantum yield of fluorescent nanoparticles are ideal for long-term monitoring of molecular events in living organisms. Here, we discuss delivery of three fluorescent nanoparticles in A549 cells, rotifers, Gram-negative bacteria, Gram-positive bacteria, and archaea. As these nanoparticles cannot enter cells, arginine-rich cell-penetrating peptides (CPPs) were used to enhance their internalization at the cellular or organismal level. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and sulforhodamine B (SRB) assay demonstrated that CPP complexed fluorescent nanoparticles did not produce lethal effect in all organisms tested. The discussion of these nanomaterials in this chapter intends to broaden our understanding

*Betty Revon Liu, Yue-Wern Huang and Han-Jung Lee*

of their biocompatibility in organisms of various hierarchical levels.

**Keywords:** fluorescent nanoparticles, cell-penetrating peptides, hypotoxicity, rotifer,

Nanoscience and nanotechnology are fast growing multidisciplinary fields in the past two decades [1]. Nanomaterials are the foundation of devices and systems in various industries that revolutionize functionalities of end-user products [2]. Nanomaterials range from simple zero-dimensional structures such as nanodots [3], wire-like nanocomposites in one-dimension nanoscales [4], to two-dimensional nanosheets, and to three-dimensional structures [5, 6]. Furthermore, anisotropy and unique nano level physical and chemical properties can result in nanomaterials of the same elemental compositions having totally different functionalities [6, 7].

#### **Chapter 4**

*Biotechnology and Bioengineering*

[21] Njoku CO, Alafiatayo

NVRI, Vom. 1984

142, 146

[20] Sofowora A. Medicinal Plants and Traditional in Africa. Part 11. Ife, Nigeria: Pitman Pess Ltd; 1984. p. 128,

RA. Comparative pathology of the main bovine skin disease in Nigeria. In: A paper presented at the National Conference on Disease of Ruminants,

[22] Ames BN, Profet M, Gold LS. Nature's chemicals and synthetic chemicals. Comparative Toxicology

PNAS. 1990;**87**:7782-7786

**32**
