**6. Host specificity of the plant growth-promoting cooperation**

Although plants harbor a diverse community of fungi, a preferential interaction exists between certain PGPF and a particular host. Once a particular host mutualizes this fungus, it undergoes host-specific adaptations. The outcome of such adaptations is a highly specialized and finely tuned mutualism, leading to improved responsiveness to each other needs. Evidences show that PGPF that induce growth in one plant species do not necessarily have the same effect in other species [5]. Some PGPF exert general growth promotion effects in several plant species, other fungi only do so in specific host plant. A field study showed that most of eight non-sporulating PGPF isolates enhanced the growth of one wheat variety, whereas a few isolates enhanced the growth of the other variety [87]. Moreover, at least four isolates increased yields of both varieties. Thus, the efficacy of the PGPF isolates depended upon the wheat variety in addition to their inherent growth promoting abilities. Similarly, many of the zoysiagrass PGPF isolates promoted growth of bentgrass [4], in contrast to a few isolates enhanced growth in soybean [88]. Similarly, nine isolates belonging to *Phoma* sp. and one non-sporulating fungus caused consistent plant length enhancement in cucumber cv. Shogoin fusiharii compared to nine isolates except the nonsporulating fungus in cv. Aodai Kyuri. Again, plant length enhancement in cv. Jibai was shown by eight *Phoma* sp. and one non-sporulating fungus compared to five *Phoma* sp. isolates in cv. Ociai fushinari [62]. Identically, *Pe. simplicissimum* GP17-2 and *F. equiseti* 19–1 demonstrated sufficient growth-promoting effects on different host plants [4, 9, 60], but did not have effect on *Lotus japonicas* [89]. The outcome of the plant-PGPF interaction, therefore, depends on the plant and PGPF species. It is likely that the specific interaction develops during long-term co-evolution, as it has been observed for compatible and incompatible interactions of pathogens with plants [90]. Moreover, certain components of root exudates may attract and interact microbe specifically and allow it colonize the roots.

### **7. Mechanisms of plant growth promotion**

The course of plant growth promotion by PGPF is complex and often cannot be attributed to a single mechanism. Various mechanisms that are known to modulate plant growth and development can be either direct or indirect. Direct growth promotion occurs when substances produced by the fungi or nutrient available by them facilitate plant growth. On the other hand, the ability of fungi to suppress plant pathogens and to ameliorate stress are considered major indirect mechanisms of plant growth promotion by PGPF. A particular PGPF may affect growth and development of plants using one or more of these mechanisms (**Table 3**).

#### **7.1 Phosphate solubilization**

Phosphorus is the second most important and frequently limiting macronutrient for plant growth and productivity. It is an important component of the key macromolecules in living cells and thereby, required for wide array of functions necessary for the survival and growth of living organisms. Despite the abundance of phosphorus in agricultural soils, the majority occurs in an insoluble form. Phosphorus forms complex compounds by reacting with iron, aluminum or calcium depending on the soil types and becomes insoluble and unavailable to plants [102]. To circumvent this problem, phosphate-solubilizing PGPF can play an important role dissolving insoluble P into the soluble form and making it available for plants. PGPF produce

*Organic Agriculture*

improve the plasticity of complex plant traits.

great promise in the improvement of agriculture yields.

**5. Duration of sustained plant growth promotion effect by PGPF**

The duration of biofunctional activities of PGPF in plants is a key factor for their effective application in the field. Naturally, a legitimate question may arise whether PGPF isolates that have shown promising effects on early growth stage of plants, could also affect the middle or late ontogenetic stages and ultimately contribute to yield increases at harvest. As for potato, an increase in leaf, shoot, and tuber weight was observed by a nonpathogenic isolate (No. 521, AG-4) of *Rh. solani* 63–70 days after planting, while it was not expressed in yield at harvest [85]. Conversely, increased growth responses of wheat plants treated with PGPF were observed during seedling (2 weeks after sowing), vegetative (4 weeks), preflowering (6 weeks), flowering (10 weeks) and seed maturation stages (14 weeks) [4]. The isolates of *Phoma* sp. (GS6-1, GS7-4) and non-sporulating fungus (GU23- 3), increased plant height, ear-head length and weight, seed number and plant biomass at harvest [79]. Again, isolates of *Phoma* sp. and non-sporulating fungus significantly increased plant length, dry biomass, leaf number and fruit number of cucumber cv. Jibai until 10 weeks post planting in greenhouse trials [62]. These isolates were equally effective in promoting growth and increasing yield of cucumber at 6 and 10 weeks post planting in the field [62]. There are other PGPF, which as well have shown the ability to confer long-term growth benefits to different plants. Rice and pea plants inoculated with *Westerdykella aurantiaca* FNBR-3, *T. longibrachiatum* FNBR-6, *Lasiodiplodia* sp. FNBR-13 and *Rhizopus delemar* FNBR-19 showed a stimulatory increase of growth for 8 weeks in the greenhouse [86]. Similarly, a single inoculation with inoculum of *Penicillium* and *Pochonia* affected the whole life cycle of tomato and Arabidopsis, respectively, accelerating the growth rate, shortening their vegetative period and enhancing seed maturation [34, 81]. As such, majority of PGPF strains are able to induce sustained beneficial effects on plant growth. The basis of sustained effects of PGPF on plants is not fully understood. One possibility is that the fungus continues to colonize the root system and establishes a life-long colonization with crop roots. The ability of PGPF to confer sustained benefit to plant is of great agriculture importance in terms of

**4.6 Impact of PGPF on yield**

increase the availability of the land for subsequent cropping. This indicates that PGPF

PGPF show promising ability to promote growth through extensive improvements and betterment of fundamental processes operating in the plants, all of which directly and indirectly contributes to the crop yield increase. Inoculation of banana (cv. Giant Cavendish and Grand Nain) with *F. oxysporum* resulted in 20–36% yield increase in the field [29]. Soil treatment with *T. harzianum* alone or in combination with organic amendment and fungicide significantly improved seed yield in pea [83] and chickpea [58]. Similarly, soil treatment with *T. viride* produced significantly the highest number of fruits per plant, number of seeds per fruit, fruit weight and dry weight of 100 seeds as compared to untreated control [84]. The beneficial association of plants with nonpathogenic binucleate *Rhizoctonia* spp. resulted in increase in yield of carrot, lettuce, cucumber, cotton, radish, wheat, tomato, Chinese mustard and potato [13, 45, 46]. These results demonstrate that PGPF hold

**74**

improving crop yield.

**77**

**Table 3.**

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation*

Suppressed damping off caused by *Pythium irregular, Pythium* sp*., Pythium paroecandrum, Pythium aphanidermatum*

Induced systemic resistance against *Colletotrichum graminicola*

Suppressed bacterial wilt disease caused

Suppressed Fusarium wilt caused by *Fusarium oxysporum* f. sp. *ciceris*

Suppressed damping off caused by

Suppressed nematodes *Pratylenchus goodeyi* and *Helicotylenchus multicinctus*

Suppressed seedling mortality by

Mitigation of oxidative stress due to

Minimized Cu-induced electrolytic leakage and lipid peroxidation

Produced abundant classes of VOCs (sesquiterpenes and diterpenes)

Produced 2-methyl-propanol and

Produced abundant amount of isobutyl alcohol, isopentyl alcohol, and

including β-caryophyllene

3-methyl-butanol

3-methylbutanal

Produced mainly terpenoid-like volatiles

Enhanced maize seedling copper stress

*Rhizoctonia solani* AG4

*Rhizoctonia solani*

NaOCl and cold stress

tolerance

Suppressed *Fusarium graminearum Sphaerodes*

Increased tolerance to salt stress *T. harzianum* T-22 [69]

Increased tolerance to drought stress *T. atroviride ID20G* [99]

and *Rhizoctonia solani* AG4

by *Ralstonia solanacearum*

**Mechanisms Specific activities PGPF strain References**

Sterile fungus GSP102, *T. harzianum* GT3-2, *F. equiseti* GF19-1, *Pe. simplicissimum* GP17-2

*T. harzianum* TriH\_ JSB27*, Phoma multirostrata* PhoM\_ JSB17*, T. harzianum* TriH\_ JSB36*, Pe. chrysogenum* PenC\_ JSB41

*mycoparasitica*

*F. oxysporum*V5W2, Eny 7.11o and Emb 2.4o

*T. harzianum* Rifai strain 1295-22

*F. oxysporum* NRRL 26379, NRRL 38335

FS2

*Talaromyces wortmannii*

*T. harzianum* T22 [78]

*T. harzianum* T-75 [58]

*Pe. viridicatum* GP15-1 [35]

*T. harzianum* isolate T-3 [83]

*Chaetomium globosum* [97]

*Pe. funiculosum* LHL06 [98]

*Phoma* sp. GS8-3 [100]

*T. viride* [101]

[4]

[34]

[12]

[29]

[96]

[28]

[40]

phosphate-solubilizing enzymes such as phytases and phosphatases and organic acids, which liberate P from insoluble phosphates. The most efficient phytase and phosphatase producing PGPF belong to the genera *Aspergillus, Trichoderma,* and *Penicillium* [103]. The order in terms of phytate hydrolysis efficacy was *Aspergillus* > *Penicillium* > *Trichoderma* [104]. *Fusarium verticillioides* RK01 and *Humicola* sp. KNU01 solubilized phosphate by increasing activities of acid phosphatase and alkaline phosphatase, and promoted soybean growth significantly [30]. The phosphate solubilizing fungi possess greater phosphorus solubilization ability than bacteria,

*Different mechanisms of plant growth promotion used by various plant growth promoting fungi (PGPF).*

*DOI: http://dx.doi.org/10.5772/intechopen.92338*

Suppression of deleterious pathogens

Amelioration of abiotic stress

Volatile organic compounds (VOCs)

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation DOI: http://dx.doi.org/10.5772/intechopen.92338*

#### **Table 3.**

*Organic Agriculture*

Phosphate solubilization

Mineralization of organic substrate

Phytohormone and enzyme production

**Mechanisms Specific activities PGPF strain References**

Solubilized P by organic acid activities *Pe. oxalicum* NJDL-

*F. verticillioides* RK01, *Humicola* sp. KNU01

*A. niger* BHUAS01, *Pe. citrinum* BHUPC01, *T. harzinum*

03, *Aspergillus niger* NJDL-12

*T. harzianum* GT2-1, *T. harzianum* GT3-1

*Phoma* sp.GS6-1, GS6- 2, GS7-3, GS7-4, GS8-6, GS10-1, GS10-2, sterile fungus GU23-3

*T. harzianum* Rifai

*Phoma* sp. GS8-1, GS8- 2, GS8-3, Sterile fungus

*T. harzianum* strain

*T. harzianum* strain

*globosum*CAC-1G

TK-2-4

*T. viride* [73]

*T. virensGv. 29-8* [48]

*T. harzianum* [95]

1295-22

GU21-1

T-203

Th 37

Gibberellins (GA1 and GA4) production *A. fumigatus* HK-5-2 [65] GAs production *Pe. resedanum* LK6 [39] GAs production *Penicillium* sp. Sj-2-2 [38] GAs production *Cladosporium* sp.MH-6 [24] GAs production *Pe. citrinum* IR-3-3 [37]

GAs production *Exophiala* sp. *LHL08* [26]

GAs production *A. fumigatus* HK-5-2 [65] GAs production *A. fumigatus* LH02 [18] IAA production *T. harzianum* T-22 [64]

GAs and IAA production *Chaetomium*

GAs production *Phoma herbarum*

*A. niger* 1B and 6A [56]

*A. niger* NCIM [76]

*Pe. bilaiae* RS7B-SD1 [92]

[30]

[16]

[91]

[4]

[87]

[93]

[62]

[47]

[94]

[23]

[41]

Solubilized P by acid phosphatase and

Solubilized P from rock phosphate and

Solubilize P from tricalcium phosphate

Phytase-mediated improvement in

Increased HCO3-extractable P (23%

Increased production of NH4-N and

Increased availability of ammonium nitrogen from barley grain

Solubilize minerals such as MnO2 and

Increased availability of ammonium nitrogen from barley grain

Increased concentration of Cu, P, Fe,

Increased concentration of Zn, P and

Increased soil organic carbon, N, P and

Increased availability of macro and micronutrients and organic carbon

Auxin-related compounds (indole-3-

Zn, Mn and Na in roots

alkaline phosphatase

Ca-P by organic acid

phytate phosphorus

increase)

NO2-N in soil

metallic zinc

Mn in shoot

K content

acetic acid, IAA)

Zeatin (Ze), IAA,

(ACC)

1-aminocyclopropane-1-carboxylic acid

(TCP)

**76**

*Different mechanisms of plant growth promotion used by various plant growth promoting fungi (PGPF).*

phosphate-solubilizing enzymes such as phytases and phosphatases and organic acids, which liberate P from insoluble phosphates. The most efficient phytase and phosphatase producing PGPF belong to the genera *Aspergillus, Trichoderma,* and *Penicillium* [103]. The order in terms of phytate hydrolysis efficacy was *Aspergillus* > *Penicillium* > *Trichoderma* [104]. *Fusarium verticillioides* RK01 and *Humicola* sp. KNU01 solubilized phosphate by increasing activities of acid phosphatase and alkaline phosphatase, and promoted soybean growth significantly [30]. The phosphate solubilizing fungi possess greater phosphorus solubilization ability than bacteria,

especially under acidic soil conditions [105]. The main reason is most fungi are eosinophilic, and have relatively higher growth in acidic environments than bacteria [106]. The acidity has significant influence on organic acid-mediated phosphate solubilizing activities of *Pe. oxalicum* NJDL-03 and *A. niger* NJDL-12 [91]. However, acidification is not always the major mechanism of P solubilization by *T. harzianum* Rifai 1295-22 (T-22), where pH of cultures never fell below 5.0 and no organic acids were detected [93]. Some of the reported PGPF such as *Aspergillus niger* has twin abilities of P mineralization and solubilization [104]. The fungus releases P both from organic and inorganic sources. These suggests that specific PGPF may have specific activity in solubilizing phosphate and making it available for crop growth.

#### **7.2 Substrate degradation (mineralization)**

Microorganisms primarily mediate soil nutrient pathways. Microbial mineralization of nutrients from organic matter is crucial for plant growth. Some PGPF promote plant growth, but do not produce plant hormones or solubilize fixed phosphate. Among *Pe. radicum*, *Pe. bilaiae* (strain RS7B-SD1) and *Penicillium* sp. strain KC6-W2, the strongest growth promotion in wheat, medic, and lentil was shown by *Penicillium* sp. KC6-W2, while the only significant P increase (~23% increase) was found in *Pe. bilaiae* RS7B-SD1-treated plants [92]. Similarly, seven *Trichoderma* isolates significantly improved the growth of bean seedlings; despite some of them do not possess any of the assessed growth-promoting traits such as soluble P, indole acetic acid (IAA) and siderophores [107]. These PGPF are believed to encourage plant growth by accelerating mineralization in the soil. Fungi have better substrate assimilation efficiency than any other microbes and are able to break down complex polyaromatic compounds such lignin and humic or phenolic acids [108]. A close relationship was found between the cellulose and starch degradation activity of PGPF for decomposing barley grain and their subsequent growth promotion effect in plants [109]. Application of *T. harzianum* strain Th 37 increased the availability of macro and micronutrients and organic carbonate in the ratoon initiation stage in sugarcane [94]. Colonization of *T. harzianum* in cucumber roots enhanced the availability and uptake of nutrients by the plants [47]. Cucumber plants grew better and produced more marketable fruits due to an increase in soil nutrients caused by PGPF, and accumulated more inorganic minerals like Ca, Mg, and K in aerial shoots [62]. PGPF are also directly involved the degradation of the nitrogenous organic materials through ammonization and nitrification. Formation of NH4-N and NO2-N in soil was accelerated during soil amendment with PGPF-infested barley grains [109]. More interestingly, the fungal entomopathogen *Metarhizium robertsii,* when established as a root endophyte, was shown to translocate nitrogen from a dead insect to a common bean plant host, suggesting this PGPF's potential to acquire mineral nutrients from organic matter and promote plant growth [54]. Nutrient release by mineralization could explain why PGPF other than mycorrhizae improve plant growth when added to soil.

#### **7.3 Phytohormone production**

Phytohormones are involved in many forms of plant-microbe interactions and also in the beneficial interactions of plants with PGPF. The commonly recognized classes of phytohormones produced by PGPF are the auxins (IAA) and gibberellins (GAs) (**Table 3**). IAA, the most studied auxin, regulates many aspects of plant growth, in particular, root morphology by inhibiting root elongation, increasing lateral root production, and inducing adventitious roots [48]. The *T. harzianum* T-22-mediated root biomass production and root hair development in maize is

**79**

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation*

believed to operate through a classical IAA response pathway [78]. Similarly, a direct correlation exists between increased levels of fungal IAA and lateral root

GAs are well known for their role in various developmental processes in plants, including stem elongation. Shoot elongation of waito-c rice seedlings by culture filtrates of *Pe. citrinum* IR-3-3 and *A. clavatus* Y2H0002 was attributed to the activity of physiologically active GAs existing in the culture filtrates [19, 37]. Biochemical analyses of *Penicillium* sp. LWL3 and *Pe. glomerata* LWL2 culture filtrates that enhanced the growth of Dongjiin beyo rice cultivar and in GA-deficient mutant *Waito-C* revealed the presence of IAA and various GAs [110]. Similarly, production of bioactive GAs correlated with enhanced growth of *Waito-C* under salinity by *Penicillium* sp. Sj-2-2 [38]. GA also played key roles during root coloni-

Another phytohormone through which PGPF mediate plant growth is cytokinin, especially the Zeatin. Zeatin production has been documented in *Piriformospora indica, T. harzianum* and *Phoma* sp., and the fungi that also produce other phytohormones [95, 112, 113]. *P. indica* produces low amounts of auxins, but high levels of cytokinins. *Trans*-Zeatin cytokinin biosynthesis was found crucial for *P. indica*-mediated growth stimulation in Arabidopsis [112]. This evidence suggests that PGPF often mediate the various growth and developmental processes in plants

PGPF produces a crucial enzyme ACC (1-aminocyclopropane-1-carboxylic acid) deaminase. ACC deaminase cleaves the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC), into NH3 (ammonia) and

of the microbes, but also play additional roles in the rhizosphere.

The key indirect mechanism of PGPF-mediated plant growth promotion is through their activities as biocontrol agents. PGPF protect and empower plants to resist harmful pathogens and ensure their better growth. The mechanisms by which PGPF suppress growth or activity of invading pathogens in crop plants

**7.5 Suppression of deleterious microorganisms by PGPF**

α-ketobutyrate [114]. The ACC deaminase regulates the plant growth by cleaving ACC produced by plants and thereby minimizing the ethylene level in the plant, which when present in high concentrations can lead to a reduced plant growth [115]. ACC deaminase is an inducible enzyme encoded by *acdS* genes of fungi and bacteria [116]. ACC deaminase appears to be central to the functional interactions of some plant-PGPF. *T. asperellum* T203 produced high levels of ACC deaminase and showed an average 3.5-fold induction of the *acds* gene [117]. When ACC deaminase expression is impaired in the fungus *T. asperellum* T203, the plant growth promotion abilities of this organism are also decreased [51]. The root colonizing bacteria *T. harzianum* T22 no longer promote canola root elongation after its *acdS* gene is knocked out [64]. Production of ACC deaminase was reported in some other fungi, which include *Issatchenkia occidentalis* [118], and *Penicillium citrinum* and a stramnopile, *Phytophthora sojae* [119, 120]. The ACC deaminase-producing microbes have competitive advantages in the rhizosphere over nonproducing microorganisms because the enzyme acts as a nitrogen source for them [116]. Moreover, bacteria and fungi that express ACC deaminase can lower the impact of a range of different stresses that affect plant growth and development [114]. These show that ACC deaminase is not only related to plant growth promotion abilities

development in *Arabidopsis* seedlings inoculated with *T. virens* [48].

*DOI: http://dx.doi.org/10.5772/intechopen.92338*

zation by *P. indica* in pea roots [111].

**7.4 Microbial ACC deaminase**

by influencing the balance of various plant hormones.

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation DOI: http://dx.doi.org/10.5772/intechopen.92338*

believed to operate through a classical IAA response pathway [78]. Similarly, a direct correlation exists between increased levels of fungal IAA and lateral root development in *Arabidopsis* seedlings inoculated with *T. virens* [48].

GAs are well known for their role in various developmental processes in plants, including stem elongation. Shoot elongation of waito-c rice seedlings by culture filtrates of *Pe. citrinum* IR-3-3 and *A. clavatus* Y2H0002 was attributed to the activity of physiologically active GAs existing in the culture filtrates [19, 37]. Biochemical analyses of *Penicillium* sp. LWL3 and *Pe. glomerata* LWL2 culture filtrates that enhanced the growth of Dongjiin beyo rice cultivar and in GA-deficient mutant *Waito-C* revealed the presence of IAA and various GAs [110]. Similarly, production of bioactive GAs correlated with enhanced growth of *Waito-C* under salinity by *Penicillium* sp. Sj-2-2 [38]. GA also played key roles during root colonization by *P. indica* in pea roots [111].

Another phytohormone through which PGPF mediate plant growth is cytokinin, especially the Zeatin. Zeatin production has been documented in *Piriformospora indica, T. harzianum* and *Phoma* sp., and the fungi that also produce other phytohormones [95, 112, 113]. *P. indica* produces low amounts of auxins, but high levels of cytokinins. *Trans*-Zeatin cytokinin biosynthesis was found crucial for *P. indica*-mediated growth stimulation in Arabidopsis [112]. This evidence suggests that PGPF often mediate the various growth and developmental processes in plants by influencing the balance of various plant hormones.

#### **7.4 Microbial ACC deaminase**

*Organic Agriculture*

**7.2 Substrate degradation (mineralization)**

especially under acidic soil conditions [105]. The main reason is most fungi are eosinophilic, and have relatively higher growth in acidic environments than bacteria [106]. The acidity has significant influence on organic acid-mediated phosphate solubilizing activities of *Pe. oxalicum* NJDL-03 and *A. niger* NJDL-12 [91]. However, acidification is not always the major mechanism of P solubilization by *T. harzianum* Rifai 1295-22 (T-22), where pH of cultures never fell below 5.0 and no organic acids were detected [93]. Some of the reported PGPF such as *Aspergillus niger* has twin abilities of P mineralization and solubilization [104]. The fungus releases P both from organic and inorganic sources. These suggests that specific PGPF may have specific activity in solubilizing phosphate and making it available for crop growth.

Microorganisms primarily mediate soil nutrient pathways. Microbial mineralization of nutrients from organic matter is crucial for plant growth. Some PGPF promote plant growth, but do not produce plant hormones or solubilize fixed phosphate. Among *Pe. radicum*, *Pe. bilaiae* (strain RS7B-SD1) and *Penicillium* sp. strain KC6-W2, the strongest growth promotion in wheat, medic, and lentil was shown by *Penicillium* sp. KC6-W2, while the only significant P increase (~23% increase) was found in *Pe. bilaiae* RS7B-SD1-treated plants [92]. Similarly, seven *Trichoderma* isolates significantly improved the growth of bean seedlings; despite some of them do not possess any of the assessed growth-promoting traits such as soluble P, indole acetic acid (IAA) and siderophores [107]. These PGPF are believed to encourage plant growth by accelerating mineralization in the soil. Fungi have better substrate assimilation efficiency than any other microbes and are able to break down complex polyaromatic compounds such lignin and humic or phenolic acids [108]. A close relationship was found between the cellulose and starch degradation activity of PGPF for decomposing barley grain and their subsequent growth promotion effect in plants [109]. Application of *T. harzianum* strain Th 37 increased the availability of macro and micronutrients and organic carbonate in the ratoon initiation stage in sugarcane [94]. Colonization of *T. harzianum* in cucumber roots enhanced the availability and uptake of nutrients by the plants [47]. Cucumber plants grew better and produced more marketable fruits due to an increase in soil nutrients caused by PGPF, and accumulated more inorganic minerals like Ca, Mg, and K in aerial shoots [62]. PGPF are also directly involved the degradation of the nitrogenous organic materials through ammonization and nitrification. Formation of NH4-N and NO2-N in soil was accelerated during soil amendment with PGPF-infested barley grains [109]. More interestingly, the fungal entomopathogen *Metarhizium robertsii,* when established as a root endophyte, was shown to translocate nitrogen from a dead insect to a common bean plant host, suggesting this PGPF's potential to acquire mineral nutrients from organic matter and promote plant growth [54]. Nutrient release by mineralization could explain why

PGPF other than mycorrhizae improve plant growth when added to soil.

Phytohormones are involved in many forms of plant-microbe interactions and also in the beneficial interactions of plants with PGPF. The commonly recognized classes of phytohormones produced by PGPF are the auxins (IAA) and gibberellins (GAs) (**Table 3**). IAA, the most studied auxin, regulates many aspects of plant growth, in particular, root morphology by inhibiting root elongation, increasing lateral root production, and inducing adventitious roots [48]. The *T. harzianum* T-22-mediated root biomass production and root hair development in maize is

**78**

**7.3 Phytohormone production**

PGPF produces a crucial enzyme ACC (1-aminocyclopropane-1-carboxylic acid) deaminase. ACC deaminase cleaves the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC), into NH3 (ammonia) and α-ketobutyrate [114]. The ACC deaminase regulates the plant growth by cleaving ACC produced by plants and thereby minimizing the ethylene level in the plant, which when present in high concentrations can lead to a reduced plant growth [115]. ACC deaminase is an inducible enzyme encoded by *acdS* genes of fungi and bacteria [116]. ACC deaminase appears to be central to the functional interactions of some plant-PGPF. *T. asperellum* T203 produced high levels of ACC deaminase and showed an average 3.5-fold induction of the *acds* gene [117]. When ACC deaminase expression is impaired in the fungus *T. asperellum* T203, the plant growth promotion abilities of this organism are also decreased [51]. The root colonizing bacteria *T. harzianum* T22 no longer promote canola root elongation after its *acdS* gene is knocked out [64]. Production of ACC deaminase was reported in some other fungi, which include *Issatchenkia occidentalis* [118], and *Penicillium citrinum* and a stramnopile, *Phytophthora sojae* [119, 120]. The ACC deaminase-producing microbes have competitive advantages in the rhizosphere over nonproducing microorganisms because the enzyme acts as a nitrogen source for them [116]. Moreover, bacteria and fungi that express ACC deaminase can lower the impact of a range of different stresses that affect plant growth and development [114]. These show that ACC deaminase is not only related to plant growth promotion abilities of the microbes, but also play additional roles in the rhizosphere.

#### **7.5 Suppression of deleterious microorganisms by PGPF**

The key indirect mechanism of PGPF-mediated plant growth promotion is through their activities as biocontrol agents. PGPF protect and empower plants to resist harmful pathogens and ensure their better growth. The mechanisms by which PGPF suppress growth or activity of invading pathogens in crop plants

include antibiosis, competition for nutrient and space, mycoparasitism and induced systemic resistance (ISR) [121]. PGPF of diverse genera promoted growth of fieldsoil grown cucumber by counteracting damping off pathogen *Pythium* sp. through microbial antagonism [4]. Banana plants inoculated with PGPF *F. oxysporum* significantly suppressed nematode pathogens *Pratylenchus goodeyi* and *Helicotylenchus multicinctus* resulting in up to ~20 to 36% increase in banana yields [29]. The mycoparasite *Sphaerodes retispora* has been reported to improve the plant dry weight and to decrease plant mortality in the presence of *F. oxysporum* [122]. Similarly, under phytotron conditions, seed germination, root biomass, total biomass, root length, and total length of *F. graminearum*-infected wheat were noticeably increased with the treatments of *S. mycoparasitica* and *T. harzianum*, as compared to inoculation with *F. graminearum* alone. Both mycoparasites prevented colonization and reduction in root growth by the pathogen [12]. PGPF compete with the pathogen for colonization niche on roots [79]. Other mechanisms of disease suppression by PGPF are, therefore, likely to include competition with pathogens for infection sites on the root surface. Moreover, there is a long and growing list of PGPF such as *Trichoderma, Penicillium, Fusarium, Phoma*, and non-sporulating fungi, which can protect crop plants against pathogens by eliciting ISR [14, 31, 123, 124]. Although many fungal strains to act as PGPF and elicit ISR, it is not clear how far both mechanisms are connected. These microbes may use some of the same mechanisms to promote plant growth and control plant pathogens.

#### **7.6 Rhizoremediation and stress control**

The microbial association of plants has a major influence on plant adaptation to abiotic stresses such as salinity, drought, heavy metal toxicity, extreme temperatures and oxidative stress. Recent studies indicate that fitness benefits conferred by certain PGPF contribute plant adaption to stresses [125]. There are reports of enhanced plant growth because of the association of PGPF with plants, even when plants are under suboptimal conditions [126]. Root colonization by *T. atroviride* ID20G increased fresh and dry weight of maize roots under drought stress [99]. Supplementation of *T. harzianum* to NaCl treated mustard seedlings showed elevation by 13.8, 11.8, and 16.7% in shoot, root length and plant dry weight, respectively as compared to plants treated with NaCl (200 mM) alone [127]. The fungus *Pe. funiculosum* significantly increased the plant biomass, root physiology and nutrients uptake to soybean under copper stress [98]. These fungi have been known to produce plant growth regulators (like GAs and auxins) and extend plant tolerance to abiotic and biotic stresses [23, 125]. Recurrently, *T. harzianum* T22 has little effect upon seedling performance in tomato, however, under stress; treated seeds germinate consistently faster and more uniformly than untreated seeds [69]. A few other fungi like *Microsphaeropsis, Mucor, Phoma, Alternaria, Peyronellaea, Steganosporium*, and *Aspergillus* are known to grow well in polluted medium and protect plants from adverse effects of metal stress [128]. There are numerous similar examples of PGPF ameliorating abiotic stresses and promoting plant growth. Despite significant differences between different stresses, cellular responses to them share common features. Enhanced resistance of PGPF-treated plants to abiotic stresses is explained partly due to higher capacity to scavenge ROS and recycle oxidized ascorbate and glutathione [99, 127]. The increase in proline content is found to be very useful in providing tolerance to these plants under stress [129]. Both enzymatic (peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, glutathione S-sransferase and gaucol peroxidase), and non-enzymatic (ascorbic acid, reduced glutathione, oxidized glutathione) antioxidants are induced by PGPF further

**81**

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation*

enhance the synthesis of these phytoconstituents and defend the plants from

Microorganisms produce various mixtures of gas-phase, carbon-based compounds called volatile organic compounds (VOCs) as part of their normal metabolism. The comparative analysis of experimental data has shown that volatile metabolites make a much greater contribution to the microbial interactions than non-volatile ones [130]. Recent studies reveal that VOC emission is indeed a common property of a wide variety of soil fungi, including PGPF. Some of these VOCs produced by PGPF exert stimulatory effects on plants. A PGPF, *Talaromyces wortmannii* emits a terpenoid-like volatile, β-caryophyllene, which significantly promoted plant growth and induced resistance in turnip [40]. The identified VOCs emitted by *Phoma* sp. GS8-3 belonged to C4-C8 hydrocarbons, where 2-methyl-propanol and 3-methyl-butanol formed the main components and promoted the growth of tobacco seedlings [100]. These two components were also extracted from PGPR [131]. On the other hand, 3-methyl-butanal has been reported from *T. viride* [101]. The other most abundant VOCs from *T. viride* were isobutyl alcohol, isopentyl alcohol, farnesene and geranylacetone. Arabidopsis cultured in petri plates in a shared atmosphere with *T. viride*, without direct physical contact was taller with more lateral roots, bigger with augmented total biomass (~45%) and earlier flowered with higher chlorophyll concentration (~58%) [101]. Moreover, volatile blends showed better growth promotion than individual compounds [132]. Volatile compounds produced by PGPF are also heavily involved in induce systemic resistance toward pathogens [100].

*DOI: http://dx.doi.org/10.5772/intechopen.92338*

**7.7 Production of volatile organic compounds (VOCs)**

**8. Pattern and process of root colonization by PGPF**

time required for maximum root colonization.

Root colonization is considered as an important strategy of PGPF for plant growth promotion. Root colonization is the ability of a fungus to survive and proliferate along growing roots in the presence of the indigenous microflora over a considerable period [35]. The fungus that colonizes plant root effectively is more rhizosphere competent than others [107]. Rhizosphere competence is a necessary condition for a fungus to be an efficient PGPF. Re-isolation frequency of the fungus from the colonized roots is an indirect measure of its root colonizing ability and thereby, its rhizosphere competence. In such studies, *Pe. simplicissimum* GP17-2 and *Pe. viridicatum* GP15-1 were re-isolated from *Arabidopsis* Col-0 roots 3 weeks after planting at high frequencies which were found to be >90% (**Figure 2**). Similarly, the re-isolation frequency of *Pe. janthinellum* GP16-2 from the roots of Col-0 plants was recorded to be, on average, 85% [33]. *Aspergillus* spp. PPA1 was re-isolated from the roots of cucumber plants at a frequency of 95–100% 3 weeks after planting [17], indicating a rapid and efficient root colonization by the PGPF. However, a slow root colonization by PGPF was also reported, as it was the case with *Phoma* sp. GS8-2, which achieved maximum colonization on cucumber roots at 10 weeks [62]. The relative growth rate of the fungi and roots seems to determine the length of

Some PGPF selectively colonize host roots and promote growth. Isolates of *Phoma* and sterile fungi showed poor ability to colonize the soybean roots and were unable to enhance the growth of soybean [79]. Similarly, *T. koningi* colonized roots and enhanced growth of *Lotus japonicas,* but *Pe. simplicissimum* and *F. equiseti* did not [89]. It was observed that *T. koningi* induced a transient and decreased level of defense gene expression in *L. japonicas* during its entry into the roots, while a

further damage [127].

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation DOI: http://dx.doi.org/10.5772/intechopen.92338*

enhance the synthesis of these phytoconstituents and defend the plants from further damage [127].

#### **7.7 Production of volatile organic compounds (VOCs)**

*Organic Agriculture*

include antibiosis, competition for nutrient and space, mycoparasitism and induced systemic resistance (ISR) [121]. PGPF of diverse genera promoted growth of fieldsoil grown cucumber by counteracting damping off pathogen *Pythium* sp. through microbial antagonism [4]. Banana plants inoculated with PGPF *F. oxysporum* significantly suppressed nematode pathogens *Pratylenchus goodeyi* and *Helicotylenchus multicinctus* resulting in up to ~20 to 36% increase in banana yields [29]. The

mycoparasite *Sphaerodes retispora* has been reported to improve the plant dry weight and to decrease plant mortality in the presence of *F. oxysporum* [122]. Similarly, under phytotron conditions, seed germination, root biomass, total biomass, root length, and total length of *F. graminearum*-infected wheat were noticeably increased with the treatments of *S. mycoparasitica* and *T. harzianum*, as compared to inoculation with *F. graminearum* alone. Both mycoparasites prevented colonization and reduction in root growth by the pathogen [12]. PGPF compete with the pathogen for colonization niche on roots [79]. Other mechanisms of disease suppression by PGPF are, therefore, likely to include competition with pathogens for infection sites on the root surface. Moreover, there is a long and growing list of PGPF such as *Trichoderma, Penicillium, Fusarium, Phoma*, and non-sporulating fungi, which can protect crop plants against pathogens by eliciting ISR [14, 31, 123, 124]. Although many fungal strains to act as PGPF and elicit ISR, it is not clear how far both mechanisms are connected. These microbes may use some of the same mechanisms

The microbial association of plants has a major influence on plant adaptation to abiotic stresses such as salinity, drought, heavy metal toxicity, extreme temperatures and oxidative stress. Recent studies indicate that fitness benefits conferred by certain PGPF contribute plant adaption to stresses [125]. There are reports of enhanced plant growth because of the association of PGPF with plants, even when plants are under suboptimal conditions [126]. Root colonization by *T. atroviride* ID20G increased fresh and dry weight of maize roots under drought stress [99]. Supplementation of *T. harzianum* to NaCl treated mustard seedlings showed elevation by 13.8, 11.8, and 16.7% in shoot, root length and plant dry weight, respectively as compared to plants treated with NaCl (200 mM) alone [127]. The fungus *Pe. funiculosum* significantly increased the plant biomass, root physiology and nutrients uptake to soybean under copper stress [98]. These fungi have been known to produce plant growth regulators (like GAs and auxins) and extend plant tolerance to abiotic and biotic stresses [23, 125]. Recurrently, *T. harzianum* T22 has little effect upon seedling performance in tomato, however, under stress; treated seeds germinate consistently faster and more uniformly than untreated seeds [69]. A few other fungi like *Microsphaeropsis, Mucor, Phoma, Alternaria, Peyronellaea, Steganosporium*, and *Aspergillus* are known to grow well in polluted medium and protect plants from adverse effects of metal stress [128]. There are numerous similar examples of PGPF ameliorating abiotic stresses and promoting plant growth. Despite significant differences between different stresses, cellular responses to them share common features. Enhanced resistance of PGPF-treated plants to abiotic stresses is explained partly due to higher capacity to scavenge ROS and recycle oxidized ascorbate and glutathione [99, 127]. The increase in proline content is found to be very useful in providing tolerance to these plants under stress [129]. Both enzymatic (peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, glutathione S-sransferase and gaucol peroxidase), and non-enzymatic (ascorbic acid, reduced glutathione, oxidized glutathione) antioxidants are induced by PGPF further

to promote plant growth and control plant pathogens.

**7.6 Rhizoremediation and stress control**

**80**

Microorganisms produce various mixtures of gas-phase, carbon-based compounds called volatile organic compounds (VOCs) as part of their normal metabolism. The comparative analysis of experimental data has shown that volatile metabolites make a much greater contribution to the microbial interactions than non-volatile ones [130]. Recent studies reveal that VOC emission is indeed a common property of a wide variety of soil fungi, including PGPF. Some of these VOCs produced by PGPF exert stimulatory effects on plants. A PGPF, *Talaromyces wortmannii* emits a terpenoid-like volatile, β-caryophyllene, which significantly promoted plant growth and induced resistance in turnip [40]. The identified VOCs emitted by *Phoma* sp. GS8-3 belonged to C4-C8 hydrocarbons, where 2-methyl-propanol and 3-methyl-butanol formed the main components and promoted the growth of tobacco seedlings [100]. These two components were also extracted from PGPR [131]. On the other hand, 3-methyl-butanal has been reported from *T. viride* [101]. The other most abundant VOCs from *T. viride* were isobutyl alcohol, isopentyl alcohol, farnesene and geranylacetone. Arabidopsis cultured in petri plates in a shared atmosphere with *T. viride*, without direct physical contact was taller with more lateral roots, bigger with augmented total biomass (~45%) and earlier flowered with higher chlorophyll concentration (~58%) [101]. Moreover, volatile blends showed better growth promotion than individual compounds [132]. Volatile compounds produced by PGPF are also heavily involved in induce systemic resistance toward pathogens [100].

### **8. Pattern and process of root colonization by PGPF**

Root colonization is considered as an important strategy of PGPF for plant growth promotion. Root colonization is the ability of a fungus to survive and proliferate along growing roots in the presence of the indigenous microflora over a considerable period [35]. The fungus that colonizes plant root effectively is more rhizosphere competent than others [107]. Rhizosphere competence is a necessary condition for a fungus to be an efficient PGPF. Re-isolation frequency of the fungus from the colonized roots is an indirect measure of its root colonizing ability and thereby, its rhizosphere competence. In such studies, *Pe. simplicissimum* GP17-2 and *Pe. viridicatum* GP15-1 were re-isolated from *Arabidopsis* Col-0 roots 3 weeks after planting at high frequencies which were found to be >90% (**Figure 2**). Similarly, the re-isolation frequency of *Pe. janthinellum* GP16-2 from the roots of Col-0 plants was recorded to be, on average, 85% [33]. *Aspergillus* spp. PPA1 was re-isolated from the roots of cucumber plants at a frequency of 95–100% 3 weeks after planting [17], indicating a rapid and efficient root colonization by the PGPF. However, a slow root colonization by PGPF was also reported, as it was the case with *Phoma* sp. GS8-2, which achieved maximum colonization on cucumber roots at 10 weeks [62]. The relative growth rate of the fungi and roots seems to determine the length of time required for maximum root colonization.

Some PGPF selectively colonize host roots and promote growth. Isolates of *Phoma* and sterile fungi showed poor ability to colonize the soybean roots and were unable to enhance the growth of soybean [79]. Similarly, *T. koningi* colonized roots and enhanced growth of *Lotus japonicas,* but *Pe. simplicissimum* and *F. equiseti* did not [89]. It was observed that *T. koningi* induced a transient and decreased level of defense gene expression in *L. japonicas* during its entry into the roots, while a

**Figure 2.**

*Re-isolation of* Penicillium simplicissimum *GP17-2 and* Penicillium viridicatum *GP15-1 at higher frequencies from colonized roots of* Arabidopsis thaliana *ecotype Col-0 3 weeks after sowing.*

stimulated expression of these genes was induced by *Pe. simplicissimum* and *F. equiseti* [89]. *T. koningi* resembles symbiotic fungi, while *Pe. simplicissimum* and *F. equiseti* act similar to fungal pathogens in activating host defense. This shows that legumes selectively avoid some PGPF and thus allow only specific PGPF to interfere.

There are also PGPF, in particular, the non-sporulating sterile fungi that lack root colonization ability, but they are able to promote growth and yield of plants [62, 133]. This indicates that root colonization is not an indispensable condition for growth promotion by all PGPF. Some chemical factor(s) produced by them might be responsible for growth promotion.

The colonization of the root system of by PGPF is not always homogenous; the density of PGPF varies in different parts of the root system. The colonization of roots by the majority of PGPF appears to be higher in the upper than in the middle and lower root parts of roots, [35, 133]. The lower part was always less colonized by PGPF, especially during first 2 weeks of colonization. This is probably due to the faster growth of the roots than of the hyphae. Moreover, the main zone of root exudation is located behind the apex [134]. However, some PGPF can keep up with root growth and colonize the entire root system [35]. Only fungi with large nutrient reserves can move to the root and along the root over larger distances [135].

Anatomical data show that PGPF may colonize root tissues internally and establish a mutualistic relationship with host. *F. equiseti* GF19-1 produced abundant hyphal growth on the root surface, formed appressoria-like structures and grew in the intercellular space, not inside the cell [31]. *T. harzianum* CECT 2413 exhibited profuse adhesion of hyphae to the tomato roots and colonized the epidermis and cortex. Intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips were also observed [136]. Hyphae of *T. koningi* penetrated the epidermis and entered the intercellular inner cortex tissues [89]. Sterile red fungus has been also demonstrated to invade the inner root regions that helped plants derive nutrients from the soil and protected roots from pathogens [137].

### **9. Formulation of PGPF**

PGPF, especially *Trichoderma*, have many success stores as plant growth promoting agents and appear to have much potential as a commercial formulation. Different organic and inorganic carrier materials have been studied for effective delivery of bioinoculants. A talc-based formulation was developed for *T. harzianum* to supply concentrated conidial biomass of the fungus with high colony forming units (CFU) and long shelf life [138]. The concentrated formulation provided an extra advantage of smaller packaging for storage and transportation, and low

**83**

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation*

product cost as compared to other carriers such as charcoal, vermiculite, sawdust and cow dung. Seed application of the formulation recorded significant increase in growth promotion in chickpea [138]. Corn and sugarcane bagasse were used as potential carriers for *Trichoderma* sp. SL2 inoculants. The corn formulation of SL2 significantly enhanced rice seedlings root length, wet weight and biomass compared to inoculum mixed with sugarcane bagasse and control [139]. A spray-dried flowable powder formulation was developed for biostimulant *Trichoderma* strains using a CO2 generating dispersant system, based on polyacrylic acid, citric acid and sodium bicarbonate, polyvinyl alcohol as adhesives and lecithin as wetting agent [140]. Hydrolytic amino acids derived from pig corpses were used in the preparation of *T. harzianum* T-E5-containing bioorganic fertilizer. The resulting bioorganic fertilizer supported higher densities of *T. harzianum* T-E5 and substantially enhanced plant growth when applied as a soil amendment [141]. A composted cattle manure-based *Trichoderma* biofertilizer was developed and tested in the field. Plots fertilized with biofertilizer had the greatest aboveground biomass of any treatment and were significantly more productive than non-amended plots and plots fertilized with any rate of organic fertilizer [142]. Effective formulation of *P. indica* was prepared in talcum powder or vermiculite with 20% moisture. The talcum-based formulations performed significantly better as bioinoculant over vermiculite-based formulations in glasshouse experiments [143]. These show the feasibility of commercial level production and applicability of different PGPF formulations for plant

Because of current concerns over the adverse effects of agrochemicals, there is a growing interest in improving our understanding of the role and application of beneficial microbes in agriculture. The plant-associated growth promoting fungi show excellent potential for wider use in sustainable agriculture as they improve plant growth and yield in an ecofriendly and cost-effective manner. However, the PGPF continue to be greatly underutilized, primarily due to some practical problems such as the inconsistency in field performance, which appears to be the greatest challenge in the development of microbial inoculants for plant growth until now and well into the future. If our understanding of complex rhizosphere environment, of the mechanisms of action of PGPF and of the practical aspects of mass production, inoculant formulation and delivery increase, more PGPF products will become available. Knowledge of multiple microbial interaction with different or complementary mode of actions is also of extreme value for development of bio-formulation.

Recent advances in biotechnological tools and reliable transformation system could be useful in engineering of the PGPF to confer improved benefits to the crop. Genetic transformation and overexpression of one or more of the plant growth promoting traits that act synergistically may lead to enhanced performance by the inoculant. Research may be required periodically in order to evaluate the genetic stability and ecological persistence of the genetically modified strain. Efforts should be strengthened to foster linkage between investigators and entrepreneurs in facili-

tating technology transfer, promotion and acceptance by end users.

*DOI: http://dx.doi.org/10.5772/intechopen.92338*

growth promotion in the field.

**10. Conclusions**

*Application and Mechanisms of Plant Growth Promoting Fungi (PGPF) for Phytostimulation DOI: http://dx.doi.org/10.5772/intechopen.92338*

product cost as compared to other carriers such as charcoal, vermiculite, sawdust and cow dung. Seed application of the formulation recorded significant increase in growth promotion in chickpea [138]. Corn and sugarcane bagasse were used as potential carriers for *Trichoderma* sp. SL2 inoculants. The corn formulation of SL2 significantly enhanced rice seedlings root length, wet weight and biomass compared to inoculum mixed with sugarcane bagasse and control [139]. A spray-dried flowable powder formulation was developed for biostimulant *Trichoderma* strains using a CO2 generating dispersant system, based on polyacrylic acid, citric acid and sodium bicarbonate, polyvinyl alcohol as adhesives and lecithin as wetting agent [140]. Hydrolytic amino acids derived from pig corpses were used in the preparation of *T. harzianum* T-E5-containing bioorganic fertilizer. The resulting bioorganic fertilizer supported higher densities of *T. harzianum* T-E5 and substantially enhanced plant growth when applied as a soil amendment [141]. A composted cattle manure-based *Trichoderma* biofertilizer was developed and tested in the field. Plots fertilized with biofertilizer had the greatest aboveground biomass of any treatment and were significantly more productive than non-amended plots and plots fertilized with any rate of organic fertilizer [142]. Effective formulation of *P. indica* was prepared in talcum powder or vermiculite with 20% moisture. The talcum-based formulations performed significantly better as bioinoculant over vermiculite-based formulations in glasshouse experiments [143]. These show the feasibility of commercial level production and applicability of different PGPF formulations for plant growth promotion in the field.

#### **10. Conclusions**

*Organic Agriculture*

**Figure 2.**

be responsible for growth promotion.

**9. Formulation of PGPF**

stimulated expression of these genes was induced by *Pe. simplicissimum* and *F. equiseti* [89]. *T. koningi* resembles symbiotic fungi, while *Pe. simplicissimum* and *F. equiseti* act similar to fungal pathogens in activating host defense. This shows that legumes selectively avoid some PGPF and thus allow only specific PGPF to interfere.

*Re-isolation of* Penicillium simplicissimum *GP17-2 and* Penicillium viridicatum *GP15-1 at higher frequencies from colonized roots of* Arabidopsis thaliana *ecotype Col-0 3 weeks after sowing.*

There are also PGPF, in particular, the non-sporulating sterile fungi that lack root colonization ability, but they are able to promote growth and yield of plants [62, 133]. This indicates that root colonization is not an indispensable condition for growth promotion by all PGPF. Some chemical factor(s) produced by them might

The colonization of the root system of by PGPF is not always homogenous; the density of PGPF varies in different parts of the root system. The colonization of roots by the majority of PGPF appears to be higher in the upper than in the middle and lower root parts of roots, [35, 133]. The lower part was always less colonized by PGPF, especially during first 2 weeks of colonization. This is probably due to the faster growth of the roots than of the hyphae. Moreover, the main zone of root exudation is located behind the apex [134]. However, some PGPF can keep up with root growth and colonize the entire root system [35]. Only fungi with large nutrient

reserves can move to the root and along the root over larger distances [135]. Anatomical data show that PGPF may colonize root tissues internally and establish a mutualistic relationship with host. *F. equiseti* GF19-1 produced abundant hyphal growth on the root surface, formed appressoria-like structures and grew in the intercellular space, not inside the cell [31]. *T. harzianum* CECT 2413 exhibited profuse adhesion of hyphae to the tomato roots and colonized the epidermis and cortex. Intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips were also observed [136]. Hyphae of *T. koningi* penetrated the epidermis and entered the intercellular inner cortex tissues [89]. Sterile red fungus has been also demonstrated to invade the inner root regions that helped plants derive nutri-

PGPF, especially *Trichoderma*, have many success stores as plant growth promoting agents and appear to have much potential as a commercial formulation. Different organic and inorganic carrier materials have been studied for effective delivery of bioinoculants. A talc-based formulation was developed for *T. harzianum* to supply concentrated conidial biomass of the fungus with high colony forming units (CFU) and long shelf life [138]. The concentrated formulation provided an extra advantage of smaller packaging for storage and transportation, and low

ents from the soil and protected roots from pathogens [137].

**82**

Because of current concerns over the adverse effects of agrochemicals, there is a growing interest in improving our understanding of the role and application of beneficial microbes in agriculture. The plant-associated growth promoting fungi show excellent potential for wider use in sustainable agriculture as they improve plant growth and yield in an ecofriendly and cost-effective manner. However, the PGPF continue to be greatly underutilized, primarily due to some practical problems such as the inconsistency in field performance, which appears to be the greatest challenge in the development of microbial inoculants for plant growth until now and well into the future. If our understanding of complex rhizosphere environment, of the mechanisms of action of PGPF and of the practical aspects of mass production, inoculant formulation and delivery increase, more PGPF products will become available. Knowledge of multiple microbial interaction with different or complementary mode of actions is also of extreme value for development of bio-formulation.

Recent advances in biotechnological tools and reliable transformation system could be useful in engineering of the PGPF to confer improved benefits to the crop. Genetic transformation and overexpression of one or more of the plant growth promoting traits that act synergistically may lead to enhanced performance by the inoculant. Research may be required periodically in order to evaluate the genetic stability and ecological persistence of the genetically modified strain. Efforts should be strengthened to foster linkage between investigators and entrepreneurs in facilitating technology transfer, promotion and acceptance by end users.

*Organic Agriculture*


**6. Examples of Orogenic belts resulting from Ampherer-subduction zones**

Ideal examples of orogens resulting from the closure of a hyperextended rift system or an immature ocean include the Alps [34], Pyrenees [136, 137], and European Variscides [138, 139]. These orogens often occurred without significant subductionrelated magmatic activity, so the original rock units before the collision were relatively well preserved [34, 43, 44, 137, 140].