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Some Aspergillus species appear to like walnuts. My question concerns the association of Penicillium and Aspergillus. No sooner does Aspergillus colonize a walnut (or some other challenging carbon source) than Penicillium seems to move in, eventually killing the Aspergillus colony.
A totally unscientific guess is that Aspergillus is a good colonizer and Penicillium a good opportunist and that this is a common pattern with these two species. Is there any science in this direction? I do recall a sort of well-known picture from an old text in which Penicillium is shown more or less strangling a species of Aspergillus. I didn't think about it much at the time.
The image attached is not very incriminating but the theme is the same. Penicillium are the green hand-like structures strewn about the clover-like Aspergillus. Foot-cell of the latter cropped.
This seems like a simple question but a quick search doesn't reveal a lot, I think in part because this would generally come up as a contamination issue.
It is hard to find any articles on the association between Penicillium and Aspergillus species, although they are both considered two of the most common mold species found indoors.
In this study, the most prevalent spore types detected in both the indoor and outdoor air samples were generally from the Penicillium/Aspergillus group [… ] these ﬁndings are qualitatively similar to those observed in other geographical locations, conﬁrming the ubiquitous nature of these fungi.
Although these genera seem to be found commonly, it also has to do with environmental factors, and the relative humidity of the area as well.
Geographical location, climate, and short-term meteorological conditions are responsible for outdoor types and levels of fungal spores.
As for the association and apparently colonization/opportunistic behaviour of the genera, it seems like there are not any associations between the two, unless perhaps environmental conditions are subject to changing, and different mold species have different moisture/temperature thresholds, although again these mold groups tend to be generalists and do well at a wider range of environmental conditions than other fungi.
A laboratory procedure was developed in which viable peanut seeds were wounded and inoculated with field soil containing natural populations of fungi, then incubated under different conditions of seed water activity and temperature. [… ] A. parasiticus colonized peanut seeds at lower temperatures than A. flavus, and cool soil temperatures relative to temperatures of aerial crop fruits might explain why A. parasiticus is found mostly in peanuts.
Other fungi, dominated by the genera Penicillium, Fusarium and Clonostachys, colonized seeds primarily at water activities and temperatures suboptimal for section Flavi species and A. niger.
Eupenicillium ochrosalmoneum frequently sporulated on the conidial heads of section Flavi species and showed specificity for these fungi. The inoculation of wounded viable peanut seeds with soil containing natural populations of fungi provides a model system for studying the infection process, the interactions among fungi and those factors important in aflatoxin formation.
Any associations with different fungi species and their different structures, including, for example, with the conidial heads of Aspergillus seem to be currently under study:
The genus Penicillium comprises species that mostly colonize plant matter. However early reports suggest that several species are capable of parasitizing Aspergillus and sporulating on the conidial heads of the host. More recently Eupenicillium ochrosalmoneum and E. cinnamopurpureum, [… ] have been observed sporulating on the heads of Aspergillus species belonging to section Flavi during the colonization of peanut seeds.
Little is known about the host specificity underlying these Aspergillus-Penicillium associations. [… ] all species spread across Aspergillus colonies by means of aerial hyphae that grew from head to head. Additional studies are required to clarify whether Eupenicillium and Penicillium species are parasitic or simply epibiotic on their hosts.
A year after the fact I found a short passage in Thom and Raper's 1945 text, Manual of the Aspergilli. At page 59 the authors relate that Aspergillus niger colonies are commonly overrun with Penicillium rugulosum which "winds its hyphae within and about the conidiophores, and fruits in a radiating series of short-stalked penicilli surrounding the heads and upper halves of the conidiophores." There is a photo illustrating this on p. 60.
Cytotoxicity of Mycotoxins
Citrinin is produced by Penicillium citrinum and other Penicillium species. This mycotoxin has been found in wheat, oats, barley, and rye. Citrinin has also been observed in ground nuts infected with Aspergillus flavus, Penicillium citrinum, and A. terre us. It has been reported in rice and corn flour. The most frequent food commodity contaminated is fruit juices. Compared to other mycotoxins, citrinin is a relatively simple molecule (see Fig. 1 ), which chemically is (3R-trans)-4,6-dihydro-3,4,5-trimethyl-6-ox -d -3H-2-benzopyran-7-carboxylic acid.
Blue mould, caused primarily by Penicillium expansum, is a major threat to the global pome fruit industry, causing multimillion-dollar losses annually. The blue mould fungus negatively affects fruit quality, thereby reducing fresh fruit consumption, and significantly contributes to food loss. P. expansum also produces an array of mycotoxins that are detrimental to human health. Management options are limited and the emergence of fungicide-resistant Penicillium spp. makes disease management difficult, therefore new approaches and tools are needed to combat blue mould in storage. This species profile comprises a comprehensive literature review of this aggressive pathogen associated with pomes (apple, pear, quince), focusing on biology, mechanisms of disease, control, genomics, and the newest developments in disease management.
Penicillium expansum Link 1809. Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass: Eurotiomycetidae, Order Eurotiales Family Trichocomaceae, Genus Penicillium, Species expansum.
A wide host range necrotrophic postharvest pathogen that requires a wound (e.g., stem pull, punctures, bruises, shoulder cracks) or natural openings (e.g., lenticel, stem end, calyx sinus) to gain ingress and infect.
Patulin, citrinin, chaetoglobosins, communesins, roquefortine C, expansolides A and B, ochratoxin A, penitrem A, rubratoxin B, and penicillic acid.
Primarily apples, European pear, Asian pear, medlar, and quince. Blue mould has also been reported on stone fruits (cherry, plum, peach), small fruits (grape, strawberry, kiwi), and hazel nut.
Blue mould initially appears as light tan to dark brown circular lesions with a defined margin between the decayed and healthy tissues. The decayed tissue is soft and watery, and blue-green spore masses appear on the decayed area, starting at the infection site and radiating outward as the decayed area ages.
Preharvest fungicides with postharvest activity and postharvest fungicides are primarily used to control decay. Orchard and packinghouse sanitation methods are also critical components of an integrated pest management strategy.
Clinical symptoms were similar between H1N1-infected patients and COVID-19 patients
Nearly 54% of COVID-19 cases and 71% of H1N1 cases were severe (Table 1). The most common underlying diseases were hypertension, diabetes mellitus, heart disease, and liver diseases, while the most common symptoms were fever, cough and diarrhoea in both COVID-19 patients and H1N1-infected patients, although these factors were not significantly different between the two groups (Table 1). The rate of positivity for SARS-CoV-2 RNA in the faeces of COVID-19 patients was 34.32%. All patients with H1N1 infection or COVID-19 were treated with antiviral drugs some were treated with glucocorticoids. The hospital stays were longer for COVID-19 patients than for H1N1-infected patients.
Different alterations in inflammation and organ function between H1N1-infected patients and COVID-19 patients
Both COVID-19 patients and H1N1-infected patients had lower blood lymphocyte counts and higher neutrophil counts than HCs (Fig. 1A). The level of CRP, an index of bacterial infection, was significantly higher in COVID-19 patients and H1N1-infected patients than in HCs, while procalcitonin, an index of bacterial and fungal infection, was higher in H1N1-infected patients than in COVID-19 patients and HCs (Fig. 1D). Additionally, compared with HCs, both H1N1-infected patients and COVID-19 patients had significant increases in the levels of IL-2, IL-6, and IL-10, suggesting that the patients were in an inflammatory state (Fig. 1C). IL-4 and TNF-α levels were higher in COVID-19 patients than in H1N1-infected patients and HCs, suggesting that COVID-19 patients experienced more inflammation. Compared with HCs, the level of albumin was lower and the levels of ALT and GGT were higher in both H1N1-infected patients and COVID-19 patients, suggesting that the patients experienced liver injury. Interestingly, AST was only higher in H1N1-infected patients and was even higher in these patients than in COVID-19 patients, suggesting that more serious liver injury might occur in H1N1-infected patients. ALP was only lower in COVID-19 patients than in HCs (Fig. 1B). Compared with the HCs, the absolute erythrocyte count in the peripheral blood was lower in both COVID-19 patients and H1N1-infected patients, while the level of haemoglobin was only lower in the COVID-19 group, suggesting that H1N1 infection, especially COVID-19, may cause erythrocyte damage (Fig. 1A). In addition, compared with the HCs, the levels of cholesterol and uric acid were lower in COVID-19 and H1N1-infected patients, while the platelet count was only lower in H1N1-infected patients. Furthermore, the platelet count and cholesterol level were lower in H1N1-infected patients than in COVID-19 patients (Fig. 1A, D).
Significantly altered (A) blood haemocytes and haemoglobin, B liver function indicators, C inflammatory cytokines, and (D) metabolic and infectious biomarkers in the blood of patients with COVID-19 (n = 67) or H1N1 (n = 35) infections compared with HCs (n = 48). *P < 0.05 **P < 0.01 and ***P < 0.001.
Fungal richness decreased in the gut of H1N1-infected patients and COVID-19 patients
Through fungal ITS sequencing, 1587 OTUs were identified, including 557 OTUs unique to HCs, 232 OTUs unique to H1N1-infected patients, and 285 OTUs unique to COVID-19 patients (Fig. 2A). The OTU richness, as reflected by the Chao1 index, was not significantly different between COVID-19 patients and H1N1-infected patients, but that in each patient group was lower than that in the HCs (Fig. 2B). The OTU diversity, as measured by the Shannon index, was similar between COVID-19 patients and HCs and was higher in both groups than in the patient group infected with H1N1 (Fig. 2C). In the PCoA plots, HCs, COVID-19 patients and H1N1-infected patients were clustered separately (Fig. 2D), indicating that their compositions were significantly different similar results were observed in non-metric multi-dimensional scaling (NMDS) plots (Fig. 2E). These conclusions were also confirmed by permutational multivariate analysis of variance (P = 0.001).
A Venn diagram, B Chao 1 index plot, C Shannon index plot, D PCoA plot, and (E) NMDS plot based on the gut fungal operational taxonomic units (OTUs) of COVID-19 patients (n = 67), H1N1-infected patients (n = 35) and HCs (n = 48). *P < 0.05 **P < 0.01 and ***P < 0.001.
Fungal dysbiosis in the guts of H1N1-infected patients and COVID-19 patients
Compared with HCs, all significant alterations in the gut mycobiota in COVID-19 patients were depletions of specific fungal taxa, such as members of Ascomycota and Basidiomycota, which was observed in both the discovery (COVID-19, n = 34 HCs, n = 23) and validation (COVID-19, n = 33 HCs, n = 23) cohorts. In the phylum Ascomycota, most of the depleted taxa belonged to Aspergillaceae, such as Penicillium citrinum, Penicillium polonicum, and Aspergillus with its five species (Fig. 3B–E). Furthermore, Candida parapsilosis, Talaromyces wortmannii, and two unclassified species that separately belonged to Didymellaceae or Onygenales were also depleted in COVID-19 patients. In the phylum Basidiomycota, five species, including Malassezia yamatoensis, Rhodotorula mucilaginosa, Moesziomyces aphidis, Trechispora sp. and Wallemia sebi, were significantly depleted (Fig. 3C–E). Similar results were observed in the phylum Mucoromycota and for the species Mucor racemosus (Fig. 3A–E). Fungal taxa, such as Candida albicans, that were altered in COVID-19 patients versus HCs in only the discovery or validation cohorts are shown in Supplementary Fig. 1.
A Phyla, B genera, and (C–E) species that were differently distributed between at least two groups (COVID-19 patients, H1N1-infected patients and HCs) in both the discovery and validation cohorts. The figure was generated by combining data from the discovery and validation cohorts (COVID-19, n = 67 H1N1, n = 32 HCs, n = 46). *P < 0.05 **P < 0.01 and ***P < 0.001 in the discovery cohort. # P < 0.05 ## P < 0.01 and ### P < 0.001 in the validation cohort.
Compared with HCs, H1N1-infected patients were mainly characterized by enrichment of the phylum Ascomycota and depletion of an unclassified fungus, which was observed in both the discovery (H1N1, n = 20 HCs, n = 30) and validation (H1N1, n = 12 HCs, n = 16) cohorts (Fig. 3A). In the phylum Ascomycota, Candida glabrata, Fusarium proliferatum, and two species belonging to Helotiales and Sordariales were enriched meanwhile, fungi such as Cladosporium, Aspergillus, Penicillium, Aspergillus niger, and Penicillium polonicum were depleted in H1N1-infected patients. In the phylum Basidiomycota, an unclassified species of Exidiaceae was enriched, while Trechispora sp., Rhodotorula mucilaginosa, Moesziomyces aphidis, and Wallemia sebi were depleted (Fig. 3B–E). Interestingly, the phylum Chlorophyta (belonging to the kingdom Plantae) and its species Trebouxia decolorans (Fig. 3A, C) as well as an unclassified species belonging to the kingdom Chromista were enriched in the gut mycobiota of H1N1-infected patients (Fig. 3D). Additionally, fungal taxa such as Saccharomyces cerevisiae that were altered in H1N1-infected patients compared with the HCs in only the discovery or validation cohorts are shown in Supplementary Fig. 2.
Compared with H1N1-infected patients, the alterations in the gut mycobiota of COVID-19 patients were mainly characterized by the depletion of the Ascomycota taxa, as observed in both the discovery (COVID-19, n = 34 H1N1, n = 20) and validation (COVID-19, n = 33 H1N1, n = 12) cohorts (Fig. 3A). The phylum Ascomycota, as well as its members Candida glabrata and Candida parapsilosis, and five unclassified species separately belonging to Helotiales, Pleosporales, Sordariales, Microscypha or Emericellopsis were depleted in COVID-19 patients (Fig. 3C–E). In the phylum Basidiomycota, Cystobasidium was enriched, while an unclassified species of Exidiaceae was depleted. Moreover, Trebouxia decolorans and an unclassified species belonging to the kingdom Chromista were also depleted in COVID-19 patients compared to H1N1-infected patients (Fig. 3C–E). Additionally, fungal taxa such as Meyerozyma guilliermondii that were altered in COVID-19 patients compared with H1N1-infected patients in only the discovery or validation cohorts are shown in Supplementary Fig. 3.
The P values of ANOSIM comparing the gut mycobiota compositions of patients with mild and severe COVID-19 (P = 0.69) and comparing those of COVID-19 patients in and out of the hospital (P = 0.93) were both greater than 0.05. This indicates that the composition of the gut mycobiota was similar in these groups and was not significantly influenced by COVID-19 severity or treatment in our cohorts.
In the receiver operating characteristic (ROC) curve analysis, when the area under the ROC curve (AUC) of both the discovery and validation cohorts higher than 0.7 (which is indicative of a discriminatory effect) was taken as a threshold, no signal fungal taxa could reliably discriminate COVID-19 patients from HCs or H1N1-infected patients. In contrast, Trebouxia decolorans could discriminate H1N1-infected patients from HCs or COVID-19 patients (Supplementary Fig. 4A). Remarkably, only Penicillium polonicum could discriminate HCs from both H1N1-infected patients and COVID-19 patients, suggesting that this species is a potential health marker of the gut mycobiota (Supplementary Fig. 4B).
Fungal burden increased in the guts of H1N1-infected patients and COVID-19 patients
The total quantity of fungi in H1N1-infected patients, COVID-19 patients or HCs was assayed by qPCR. First, we evaluated the ratio of ITS copies of fungi obtained by qPCR to the amount of fungi obtained by plate counting using Clavispora lusitaniae CICC 32908 as a reference strain. Our results showed that this ratio was 4.04 ± 0.56 (Fig. 4A). Next, we performed qPCR using the DNA extract of faecal samples. The median (25th, 75th centiles) number of fungi per gram of faeces was 2.73E+04 (3.81E+03, 9.70E+05) for COVID-19 patients, 4.93E+04 (1.72E+03, 1.35E+06) for H1N1-infected patients, and 1.29E+04 (6.03E+02, 5.43E+04) for HCs. Although it is limiting to use Clavispora lusitaniae CICC 32908 to evaluate the abovementioned ratio, the total amount of fungi in the faeces of either COVID-19 patients or H1N1-infected patients was significantly higher than that in HCs in this study with the same method (Fig. 4B). In addition, we found no significant difference in the number of gut fungi between COVID-19 patients at admission and discharge.
A The ratio of ITS copies of Clavispora lusitaniae CICC 32908 obtained by qPCR to CICC 32908 number obtained by culture. The experiment was repeated five times. B Number of fungi in one gram of faeces from the healthy controls (n = 48), COVID-19 patients (n = 67) or H1N1-infected patients (n = 35). The ITS copies in the faeces obtained by qPCR were converted to fungal numbers based on the ratio of ITS copies of Clavispora lusitaniae CICC 32908 obtained by qPCR to CICC 32908 number obtained by culture. *P < 0.05 **P < 0.01 and ***P < 0.001.
Associations between faecal mycobiota and the bacterial microbiota
In COVID-19 patients, the phylum Ascomycota and its members Aspergillus niger and Aspergillus rugulosus were negatively correlated with Lachnospiraceae and its genera Agathobacter, Dorea and Roseburia with Ruminococcaceae and its genera Butyricicoccus and Faecalibacterium and with Eggerthella and Veillonella (Fig. 5A). In contrast, the phylum Mucoromycota was positively correlated with Peptostreptococcaceae, Bifidobacterium, Fusicatenibacter and Intestinibacter, as well as Aspergillus with Agathobacter.
Spearman correlations of significantly altered gut mycobiota with significantly altered gut microbiota in (A) COVID-19 patients or (B) H1N1-infected patients. Spearman correlations of significantly altered gut mycobiota with significantly altered blood biomarkers in (C) COVID-19 patients or (D) H1N1-infected patients. Only correlations with a P < 0.05 and a correlation coefficient >0.4 or <−0.4 are shown.
In H1N1-infected patients, more correlations between the gut mycobiota and microbiota were observed, and the absolute values of their correlation coefficients were higher as well (Fig. 5B). More than half of these correlations were positive for Aspergillus and its genera with Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae, as well as their members. Interestingly, Penicillium and Penicillium polonicum were also positively correlated with Akkermansia. In contrast, there were negative correlations of Ascomycota with bacteria such as Roseburia or Marvinbryantia.
Associations of the gut mycobiota with clinical symptoms of H1N1 infection or COVID-19
In COVID-19 patients, Aspergillus niger was highly significantly positively correlated with the incidence of diarrhoea (r = 0.51, P = 8.31E−06), while Penicillium citrinum was highly significantly negatively correlated with the blood CRP level (r = −0.41, P = 9.37E−04) (Fig. 5C). Interestingly, Rhodotorula mucilaginosa was highly negatively correlated with blood angiotensin-converting enzyme (r = −0.79, P = 0.036). Furthermore, many gut fungi were positively correlated with blood immune indicators, such as the absolute number of blood lymphocytes. Remarkably, the faecal positivity rate of SARS-CoV-2 RNA was positively correlated with diarrhoea and disease severity.
In H1N1-infected patients, Aspergillus was positively correlated with the CRP level, while Mucoromycota was negatively correlated with the procalcitonin level (Fig. 5D). Furthermore, there were other positive correlations, such as those of Aspergillus penicillioides, with the serum levels of TNF-α, IL-2 and IL-10.
A wide array of signalling molecules
When bacteria and fungi encounter each other in an environmental niche, they communicate through release of specific signals. For a molecule to act as signal, it must be able to (i) diffuse from a producer, (ii) interact with a receiver, (iii) elicit a response in the receiver, and (iv) impact both the producer and the receiver in a concentration-dependent manner (Whiteley et al., 2017 ). Thus, these signals often elicit a response in the form of changes in gene expression, observed phenotype, or release of a response signal, or some combination of these responses based on the specificity of the interacting partner (Table 1). Although the nature of the signalling receptors is not fully elucidated, it is likely that receptors are key components involved in recognition of some microbial ligands (Michie et al., 2016 Brown et al., 2018 ). For example, Candida albicans recognition of lactic acid requires the G protein-coupled receptor (GPCR) Gpr1 (Ballou et al., 2016 ) and quorum molecules are recognized by various bacterial receptors (McCready et al., 2019 ).
Microbes are equipped with different types of SSMs that work together to achieve the common goal of defence and social networking. Antibiotics (broadly encompassing both anti-bacterial and anti-fungal agents) are bioactive secondary metabolites (SMs) with therapeutic activity that can kill or inhibit living organisms (Hutchings et al., 2019 ). Volatile organic compounds (VOCs) are chemically diverse, small lipophilic metabolites (Weisskopf et al., 2016 ). Quorum sensing molecules (QSMs, commonly known as autoinducers in bacteria) are synthesized in a chemical gradient and when detected by the receiving microbe, result in developmental shifts appropriate to the environment (Zhao et al., 2017 ). However, at minimal concentrations, antibiotics, VOCs and QSMs act as signalling molecules and can induce SM production, motility and biofilm formation in interacting microbes (Linares et al., 2006 Yim et al., 2007 Mitova et al., 2008 Romero et al., 2011 Effmert et al., 2012 Boedicker and Nealson 2015 van der Meij et al., 2017 ).
The signalling nature of these SSMs and their ability to induce changed gene expression highlight their involvement in inter and/or intraspecies communication. These SSMs often share a common feature of acting as weapons at higher concentrations and as communication signals at sub-inhibitory concentrations. Here, we will discuss some of the characterized signal molecules, that regulate BFIs, by grouping them in agricultural and medical contexts.
The link between fungi and severe asthma: a summary of the evidence
There is current evidence to demonstrate a close association between fungal sensitisation and asthma severity. Whether such an association is causal remains to be confirmed, but this is explored by means of a detailed literature review. There is evidence from two randomised controlled trials that, in the example of allergic bronchopulmonary aspergillosis (ABPA), treatment with systemic antifungal therapy can offer a therapeutic benefit to approximately 60% of patients. ABPA is only diagnosed if a combination of clinical and immunological criteria is achieved. It is not known whether such cases are a discrete clinical entity or part of a spectrum of the pulmonary allergic response to fungi or fungal products. This paper describes the epidemiological evidence that associates severity of asthma with fungi and discusses possible pathogenetic mechanisms. Many airborne fungi are involved, including species of Alternaria, Aspergillus, Cladosporium and Penicillium, and exposure may be indoors, outdoors or both. The potential for a therapeutic role of antifungal agents for patients with severe asthma and fungal sensitisation is also explored. Not only are many patients with severe asthma desperately disabled by their disease, but, in the UK alone, asthma accounts for 1,500 deaths per yr. The healthcare costs of these patients are enormous and any treatment option merits close scrutiny. Within this report, the case for the consideration of a new term related to this association is put forward. The current authors propose the term "severe asthma with fungal sensitisation". However, it is recognised that enhanced and precise definition of fungal sensitisation will require improvements in diagnostic testing.
Microbial Interactions and it's Types ( Mutualism, Commensalism, Amensalism, Compitition, Parasitism, Predation & Neutral association)
The microorganisms that inhabit soil exhibit many different types of association or interaction.
Microbial interaction may be positive such as mutualism, proto-cooperation, commensalism or may be negative such as parasitism, predation or competition.
Types of Microbial Interactions
There are three types of Microbial Interactions, found in soil.
1. Positive interaction :
2. Negative interaction :
3. Neutral association :
1). Positive Associations :
(a) Mutualism :
This is a symbiotic association where both the partners are benefited.
The manner in which the benefit is derived varies.
* Synergism :
* Syntrophism :
* Rhizosphere effect :
* Lichens :
* Mycorrhizae :
- The fungi penetrates the outermost layers of tree roots and grows on the outer surface of the root. The fungal mycelium forms a sheath around the root of plants.
- In this association fungi obtain nutrients from plants, and in return it gets water and minerals from the soil through fungi.
- Most of these fungi cannot be cultivated in absence of plants.
- The plant growth is adversely affected in absence of fungi.
- In this association fungi grow within the cells of plant roots.
- Sometimes the fungi form branch like structure or specialized inclusions called vesicles and arbuscules inside the plant cells and so, are called vesicular arbuscularmycorrhizae (VAM).
- VAM fungi play an important role in increasing plant growth by increasing supply of phosphorous to host plant. Also make the plant more resistant to plant diseases.
- These arbusculars are digested by the plant cells and the nutrients released from the fungi are used by the plants.
- The fungi in turn obtain nutrients from the plant tissues.
It is an association between organism in which one partner benefits, while the other partner is not affected.
(i) This occurs in soil with respect to degradation of cellulose and lignin.
e.g., association occurs between fungi and bacteria in soil.
• The cellulose degrading fungi degrades cellulose, produce glucose and organic acids which is used by bacteria for their growth.
• Thus bacteria are benefited by the association and fungi are not affected.
• Commensalism also exists when a mixed culture of organisms cause degradation of complex molecules which
cannot be done by Individual organism.
e.g., pure cultures of microorganisms cannot degrade lignin in laboratory, but the mixed microbial flora can easily degrade lignin forest soil.
• Many commensal relationships are based on the production of growth factors.
• Many nutritionally fastidious bacteria in soil often depend on growth factors such as vitamins and amino acids released from other organisms.
2). Negative associations :
(a) Amensalism / Antagonism :
(b) Competition :
• This is a relationship in which one organism lives Inside or on the surface of other organism at the expense of the other organism.
• One partner is called parasite and the other is called host.
• The parasite feeds on the cells, tissues or body fluids of host, hence it is always harmed.
• All plants, animals and microorganisms can be attacked by microbial parasites.
e.g., Parasitic association between bacteria. Bacterium Bdellovibrio bacteriovorous present In soil and sewage is a parasite of gram-negative bacteria.
All these negative associations normally control population densities in soil.
The soil fungus Artrobotrys conoides produces hyphae that form rings to trap protozoa and nematodes and digest it.
Some fungi such as Trichoderma and Lactisaria species can destroy other plant pathogenic soil fungi. Such mycoparasitic fungi are used as biopesticides to control plant diseases.
3). Neutral Associations :
It is the association in which both partner do not exhibit positive or detrimental effect on each other.
This type of association occurs when each partner can utilize different nutrients without producing end products which is inhibitory to other.
The two partners do not compete for nutrition even if they are present in low conecntrations.
Such a condition may be transitory as conditions or the relationship might change with variation in environmental conditions.
Fungi Questions and Answers | Microbiology | Biology
Frequently asked questions and answers on Fungi. In this article we will discuss about:- 1. Definition of Fungi 2. Origin of Fungi 3. Metabolism 4. Characteristics 5. Structures 6. Reproduction 7. Classification 8. Importance 9. Spore Forms 10. Laboratory Diagnosis.
- Questions and Answers # Definition of Fungi
- Questions and Answers # Origin of Fungi
- Questions and Answers # Metabolism of Fungi
- Questions and Answers # Characteristics of Fungi
- Questions and Answers # Structures of Fungi
- Questions and Answers # Reproduction of Fungi
- Questions and Answers # Classification of Fungi
- Questions and Answers # Importance of Fungi
- Questions and Answers # Spore Forms in Fungi
- Questions and Answers # Laboratory Diagnosis of Fungal Infection
Questions and Answers # 1. Definition of Fungi:
The fungi are more evolutionarily advanced forms of microorganisms, as compared to ” the prokaryotes (prions, viruses, bacteria). They are classified as eukaryotes. i.e., they have a diploid number of chromosomes and a nuclear membrane and have sterols in their plasma membrane. Genetic complexity allows morphologic complexity and thus these organisms have complex structural features that are used in speciation.
Fungi can be divided into two basic morphological forms, yeasts and hyphae. Yeasts are unicellular fungi which reproduce asexually by blastoconidia formation (budding) or fission. Hyphae are multi-cellular fungi which reproduce asexually and/or sexually. Dimorphism is the condition where by a fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions.
Very few fungi exhibit dimorphism. Most fungi occur in the hyphae form as branching, threadlike tubular filaments. These filamentous structures either lack cross walls (coenocytic) or have cross walls (septate) depending on the species. In some cases septate hyphae develop clamp connections at the septa which connect the hyphal elements.
A mass of hyphal elements is termed the mycelium (synonymous with mold). Aerial hyphae often produce asexual reproduction propagates termed conidia (synonymous with spores). Relatively large and complex conidia are termed macroconidia while the smaller and simpler conidia are termed microconidias.
When the conidia are enclosed in a sac (the sporangium), they are called endospores. The presence/absence of conidia and their size, shape and location are major features used in the laboratory to identify the species of fungus in clinical specimens.
Asexual reproduction, via conidia formation, does not involve genetic recombination between two sexual types whereas sexual reproduction does involve genetic recombination between two sexual types.
Questions and Answers # 2. Origin of Fungi:
Ireland of the 1840s was an economically depressed country of eight million people. Most were tenant farmers paying rent to landlords who were responsible, in turn, to the English owners of the property. The sole crop of Irish farmers was potatoes, grown season after season on small tracts of land. What little corn was available was usually fed to the cows and pigs. Early in the decade, heavy rains and dampness portended calamity.
Then, on August 23, 1845, The Gardener’s Chronicle and Agricultural Gazette reported:
“A fatal malady has broken out amongst the potato crop. On all sides we hear of the destruction. In Belgium, the fields are said to have been completely desolated.”
The potatoes had suffered before. There had been scab, drought, “curl,” and too much rain, but nothing was quite so destructive as this new disease. It struck down the plants like frost in the summer. Beginning as black spots, it decayed the leaves and stems, and left the potatoes a rotten, mushy mass with a peculiar and offensive odor. Even the harvested potatoes rotted.
The winter of 1845-1846 was a disaster for Ireland. Farmers left the rotten potatoes in the fields, and the disease spread. First the farmers ate the animal feed and then the animals. They also devoured the seed potatoes, leaving nothing for spring planting.
As starvation spread, the English government attempted to help by importing corn and establishing relief centers. In England, the potato disease had few repercussions because English agriculture included various grains, but in Ireland famine spread quickly.
After two years, the potato rot seemed to slacken, but in 1847 it was back with a vengeance. Despite relief efforts by the English, over two million Irish suffered death from starvation. Eventually, about 900,000 survivors set off for Canada and the United States. Those who stayed had to deal with economic and political upheaval as well as misery and death.
The potato blight faded in 1848, but did not vanish. Instead, it emerged again during wet seasons and blossomed anew. In the end, hundreds of thousands of Irish left the land and moved to cities or foreign countries. During the 1860s, great waves of Irish immigrants came to the United States. Many Americans are descended from those starving, demoralized farmers.
Such are the historic, political, economic, and sociological effects of one species of fungus. Other fungal diseases of fruits, grains, and vegetables can be equally devastating. In addition, we shall take note of several widespread human and animal diseases that are due to fungi and we shall encounter many beneficial fungi such as those used to make antibiotics, bread, and foods or used as insecticides. Our study will begin with a focus on the structures, growth patterns, and life cycles of fungi.
All fungi are free living, i.e., they are not obligate intracellular parasites. They do not contain chlorophyll and cannot synthesize macromolecules from carbon dioxide and energy derived from light rays. Therefore all fungi are heterotrophs, living on preformed organic matter.
For medical purposes the important aspects of fungal metabolism are:
1. The synthesis of chitin, a polymer of N-acetyl glucosamine, and other compounds, for use in forming the cell wall. These induce immune hypersensitivity.
2. The synthesis of ergosterol for incorporation into the plasma membrane. This makes the plasma membrane sensitive to those antimicrobial agents which either block the synthesis of ergosterol or prevent its incorporation into the membrane or bind to it, e.g. amphotericin B.
3. The synthesis of toxins such as:
(a) Ergot alkaloids – these are produced by Claviceps purpurea and cause an alpha adrenergic blockade
(b) Psychotropic agents – these include psilocybin, psilocin and lysergic acid diethylamide (LSD)
(c) Aflatoxins – these are carcinogens produced by Aspergillus flavus when growing on grain. When these grains are eaten by humans or when they are fed to dairy cattle and they get into the milk supply, they affect humans.
4. The synthesis of proteins on ribosomes that are different from those found in bacteria. This makes the fungi immune to those antimicrobial agents that are directed against the bacterial ribosome, e.g., chloramphenicol.
5. The ability of certain metabolites to alter morphology of yeast and/or be assimilated by yeast with concomitant clinical identification affects.
2. Do not contain chlorophyll
4. Absorptive heterotrophs – digest food first and then absorb it into their bodies.
5. Release digestive enzymes to break down organic material or their host.
6. Store food energy as glycogen
7. Most are saprobes – live on other dead organisms
8. Important decomposers and recyclers of nutrients in the environment
9. Most are multicellular, but some unicellular like yeast
10. Some are internal or external parasites a few are predators that capture prey
12. Lack true roots, stems, and leaves
13. Cell walls are made of chitin (a complex polysaccharide)
14. Grow as microscopic tubes or filaments called hyphae that contain cytoplasm and nuclei
15. Hyphal networks are called mycelium
17. Reproduce by sexual and asexual spores
18. Antibiotic penicillin comes from Penicillium mold
19. Classified by their sexual reproductive structures
20. Grow best in warm, moist environments preferring shade
21. Mycology – study of fungi
22. Fungicide – chemicals used to kill fungi
23. Includes yeasts, molds, mushrooms, ringworm, puffballs, rusts, smuts, etc.
24. Fungi may have evolved from prokaryotes by endosymbiosis.
1. Body of a fungus made of tiny filaments or tubes called hyphae.
2. Hyphae contain cytoplasm and nuclei and have a cell wall of chitin.
3. Each hyphae is one continuous cell.
4. Hyphae continually grow and branch.
5. Septum (septa-plural) is cross walls with pores to allow the movement of cytoplasm in hyphae.
6. Hyphae with septa are called septate hyphae.
7. Hyphae without septa are called coenocytic hyphae.
8. Tangled mats of hyphae are known as mycelium
9. All hyphae within a mycelium share the same cytoplasm so materials move quickly.
10. Hyphae grow rapidly from the tips by cell division.
11. Stolon is horizontal hyphae that connect groups of hyphae to each other.
12. Rhizoids are root like parts of hyphae that anchor the fungus.
1. Most fungi reproduce asexually and sexually.
2. Asexual reproduction produces genetically identical organisms and is the most common method used.
3. Sexual reproduction in fungi occurs when nutrients or water are scarce.
4. Fruiting bodies are modified hyphae that make asexual spores.
5. Fruiting bodies consist of an upright stalk or sporangiophore with a sac containing spores called the sporangium.
6. Types of fruiting bodies include basidia, sporangia, and ascus.
7. Spores – haploid cells with dehydrated cytoplasm and a protective coat capable of developing into new individuals.
8. Wind, animals, water, and insects spread spores.
9. When spore lands on moist surface, new hyphae form.
Questions and Answers # 6. Reproduction of Fungi:
1. Fungi reproduce asexually when environmental conditions are favorable.
2. Some unicellular fungi reproduce by mitosis.
3. Yeast cells reproduce by budding where a part of the cell pinches off to produce more yeast cells.
4. Athlete’s foot fungus reproduces by fragmentation from a small piece of mycelium.
5. Most fungi reproduce asexually by spores.
6. Penicillium mold produces spores called conidia without a protective sac on the top of a stalk called the conidiophore.
1. Fungi reproduce sexually when environmental conditions are unfavorable.
2. No male or female fungi.
3. Two mating types – plus (+) and minus (-)
4. Fertilization occurs when (+) hyphae fuse with (-) hyphae to form a 2N or diploid zygote.
5. Some fungi show dimorphism (ability to change their form in response to their environmental conditions)
Questions and Answers # 7. Classification of Fungi:
Fungi are classified by their reproductive structures. The 4 phyla of fungi are Basidiomycota, Zygomycota, Ascomycota, and Deuteromycota.
1. Called sporangium fungi or common molds.
2. Includes molds and blings such as blights such as Rhizopus stolonifer (bread mold).
3. No septa in hyphae (coenocytic).
4. Asexual reproductive structure called sporangium and produces sporangiospores.
5. Rhizoids anchor the mold, release digestive enzymes, and absorb food.
6. Asexual reproductive structure called sporangium and produces sporangiospores.
7. Sexual spore produced by conjugation when (+) hyphae and (-) fuse is called zygospore.
8. Zygospores can endure harsh environments until conditions improve and new sporangium.
2. Includes mushrooms, toadstools, puffballs, bracket fungi, shelf fungi, stinkhorns, rusts, and smuts
3. Some are used as food (mushroom) and others cause crop damage (rusts and smuts)
4. Seldom reproduce asexually
5. Basdiocarp made up of stalk called the stipe and a flattened cap.
6. Stipe may have a skirt like ring below cap called the annulus.
7. Gills are found on the underside of the cap and are lined with basidia.
8. Basidium – sexual reproductive structure that make basidiospores.
9. Basidiospores are released from the gills and germinate to form new hyphae and mycelia.
10. Vegetative structures found below ground and include rhizoids (anchor and absorb nutrients), hyphae, and mycelia.
2. Includes yeast, cup fungi, truffles, powdery mildew, and morels
1. Some are parasites causing Dutch elm disease and chestnut blight.
2. Sac Fungi can reproduce both sexually and asexually.
3. Yeast reproduces asexually by budding (form small, bud-like cells that break off and make more yeast)
4. Asexual spores called conidia form on the tips of specialized hyphae called condiophores.
5. Ascocarp – specialized hyphae formed by parent fungi during sexual reproduction.
6. Ascus – sacs within the ascocarp that form spores called ascospores.
1. Symbiotic association between a sac fungus and a photosynthetic green algae or cyanobacteria.
2. Both organisms benefit (algae makes food and fungus supplies moisture, shelter, and anchorage)
3. Grow on rocks, trees, buildings, etc. and help form soil.
4. Crustose lichens grow on rocks and trees fructose lichens grow shrub-like foliose lichens grow mat-like on the soil.
1. Symbiotic association of a fungus living on plant roots.
2. Most plants have mycorrhizae on their roots.
3. Fungus absorbs sugars made by plant.
4. Plants absorb more water and minerals with aid of the fungus.
1. Fungal spores cause allergies
2. Molds, mildew, rust, and smuts damage crops
3. Yeasts are used to make beer and bread
5. Decomposers and recyclers of nutrients
6. Mushrooms eaten as food
8. Aspergillus is used to make soy sauce
9. Cause athlete’s foot and ringworm
10. Amanita is poisonous mushroom
11. Can cause yeast infections
Species are important human pathogens that are best known for causing opportunist infections in immuno compromised hosts (e.g. transplant patients, AIDS sufferers, cancer patients). Infections are difficult to treat and can be very serious – 30-40% of systemic infections result in death.
The sequencing of the genome of C. albicans and those of several other medically relevant Candida species has provided a major impetus for Candida comparative and functional genomic analyses. These studies are aiding the development of sensitive diagnostic strategies and novel antifungal therapies.
Aspergillus spores are found nearly everywhere so we are routinely and almost constantly exposed to them. Such exposure is a normal part of the human condition and generally poses no adverse health effects. Nevertheless, Aspergillus can and does cause disease in three major ways: through the production of mycotoxins through induction of allergenic responses and through localized or systemic infections. With the latter two categories, the immune status of the host is pivotal.
Allergies and asthma are thought to be caused by an active host immune response against the presence of fungal spores or hyphae. In contrast, with invasive aspergillosis, the immune system has collapsed and little or no defence can be mounted.
The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus. Aspergillus flavus produces aflatoxin which is both a toxin and a carcinogen and which can potentially contaminate foods such as nuts. Aspergillus fumigatus and Aspergillus clavatus can cause allergic disease.
Some Aspergillus species cause disease on grain crops, especially maize, and synthesize mycotoxins including aflatoxin. Aspergillosis is the group of diseases caused by Aspergillus. The symptoms include fever, cough, chest pain or breathlessness. Usually, only patients with weakened immune systems or with other lung conditions are susceptible.
Cryptococcus neoformans can cause a severe form of meningitis and meningo­encephalitis in patients with HIV infection and AIDS. The majority of Cryptococcus species lives in the soil and do not cause disease in humans. Cryptococcus neoformans is the major human and animal pathogen.
Cryptococcus laurentii and Cryptococcus albidus have been known to occasionally cause moderate-to-severe disease in human patients with compromised immunity. Cryptococcus gattii is endemic to tropical parts of the continent of Africa and Australia and can cause disease in non-immunocompromised people
Histoplasma capsulatum can cause histoplasmosis in humans, dogs and cats. The fungus is most prevalent in the Americas, India and southeastern Asia. It is endemic in certain areas of the United States. Infection is usually due to inhaling contaminated air.
Pneumocystis jirovecii (or Pneumocystis carinii) can cause a form of pneumonia in people with weakened immune systems, such as premature children, the elderly, and AIDS patients.
Stachybotrys chartarum or “black mold” can cause respiratory damage and severe headaches. It frequently occurs in houses in regions that are chronically damp.
Questions and Answers # 9. Spore Forms in Fungi:
Fungi reproduce both asexually and sexual­ly by different types of spores. Some spores are the unit of dispersal, but others function as rest­ing spores to overcome the unfavourable period, and some others have the efficiency to serve both the above functions.
The spores are mainly divided into two groups:
The spores formed during asexual reproduction are the asexual spores. Basically the asexual spores are of two types: sporangiospores, and conidia.
The sporangiospores are developed inside a sac-like structure, the sporangium.
Sporangio­spores are of two types, zoospores and aplanospores:
Zoospores (Fig. 4.4) are the characteristic spore of the subdivision Mastigomycotina. Three types of zoo­spores are produced in this group.
i. Anteriorly uniflagellate, these are pyriform in shape having one whiplash (Synchytrium) or tinsel (Rhizidiomyces) type of flagellum anteriorly.
ii. Anteriorly biflagellate. These are pyriform in shape having two flagella (one whiplash and one tinsel type), placed anteriorly., e.g., Saprolegnia etc.
iii. Laterally biflagellate. These are kidney-shaped having two flagella (one whiplash and one tinsel type), placed laterally, e.g., Phytophthora, Pythium etc.
The sporangiospores of this type are devoid of flagella and are called aplanospores (Fig. 4.8L). It is the characteristic spore of Mucorales under Zygomycetes, e.g., Mucor, Rhizopus etc.
The conidia (Fig. 4.8A, B, C) differ from aplanospores because they are not enclosed by a separate sporangial wall. They are usually developed at the apex of hyphae.
Conidia show variability in both their form and development. They are main­ly divided on the basis of their mode of development.
There are two basic modes of development of conidia: bias- tic and thallic. In blastic type, marked enlargement of conidium initial takes place before it is delimited by the sep­tum. In thallic type, enlargement of the conidium initial takes place only after the initial has been delimited by the septum.
These are divided into two types:
1. Blastoconidia. They can develop from a cell of an undifferentiated hypha or conidiophore at o e or more points through budding, either apically or late­rally. They may develop either singly or in chains, e.g., Aureobasidium, Cladosporium etc.
2. Phialoconidia. They develop from a bottle-shaped cell, the phialide. Single conidium reaches full size before being cut off by septation and immediately another one develops underneath. Thus generally a chain of conidia are deve­loped, where younger one always remains near the mother, e.g., Tricho- derma, Fusarium.
1. Thalloconidia. In this type, the conidia are developed on hypha either singly or in short or long, branched or unbranched chains and enlargement of the conidium initial takes place only after the initial has been delimited by septum, e.g., Erysiphe, Penicillium, Geotrichum, Oidium etc.
The spores formed after sexual reproduction are the sexual spores. There are four types of sexual spores in fungi.
These are oospores, zygospores, ascospores and basidiospores:
These are diploid (2n) spores formed by the union between egg and male nucleus. Coming in contact with the oogonium, the antheridium passes its nucleus inside the oogonium through fertilisation tube and forms oospore. It is a resting spore, e.g., members of Oomycetes under Mastigomycotina.
Like Oospore, it is also a resting spore formed by the union of two gametangia into a single cell. The cell develops into a thick-walled, black and warty structure, the Zygospore, e.g., members of Zygomycetes. In majority of the members of Mastigo­mycotina and Zygomycotina, the sexually produced spores are developed freely and are not surrounded by sterile hyphae. But in Ascomycotina and Basidiomycotina, the spores are commonly surrounded by sterile hyphae and are called ascocarps and basidiocarps respec­tively.
Ascospores are developed by the union of two gametangia. The ascospores are always developed in (i.e., endogenously) a sac-like structure, the ascus, and thus the fungi containing asci are commonly called sac fungi.
The ascospores are uninucleate and uni­cellular generally oval, round or elon­gated structure. They are generally 8 in an ascus, but may be 4 (Saccharomyces cerevisiae, Saccharo­mycodes ludwigii) or numerous.
The basidiospores are developed on (i.e., exogenously) basidia. The basidia are developed from the dikaryotic cell. During this process a long gap is generally maintained between plasmogamy and karyogamy. Basidia are usually of two types: Holobasidium i.e., aseptate basidium and Phragmobasidium.
The basidiospores are unicellular, uni­nucleate, thin walled generally round to oval structure, developed on basi­dium. They are generally four in num­ber per basidium but may be two or many (e.g., Ustigo nuda tritici).
Questions and Answers # 10. Laboratory Diagnosis of Fungal Infection:
The KOH test for fungus is conducted on an outpatient basis and patients do not need to prepare in advance. Results are usually available while the patient waits or the next day if sent to a clinical laboratory. The KOH test procedure may be performed by a physician, physician assistant, medical assistant, nurse, or medical laboratory technician. If fungal cultures are required, the test is performed by a technologist who specializes in microbiology.
1. Collection – Skin, nail, or hair samples are collected from the infected area on the patient. For skin samples, a scalpel or edge of a glass slide is used to gently scrape skin scales from the infected area. For hair samples, a forceps is used to remove hair shafts and follicles from the infected site. If the test is being sent to a laboratory, the scrapings are placed in a sterile covered container.
2. The scrapings are placed directly onto a microscope slide and are covered with 10% or 20% potassium hydroxide.
3. The slide is left to stand until clear, normally between five and fifteen minutes, in order to dissolve skin cells, hair, and debris.
4. To enhance clearing dimethyl sulfoxide can be added to the slide. To make the fungi easier to see lactophenol cotton blue stain can be added.
5. The slide is gently heated to speed up the action of the KOH.
6. Adding calcofluor-white stain to the slide will cause the fungi to become fluorescent, making them easier to identify under a fluorescent microscope.
7. Place the slide under a microscope to read.
Dermatophytes are easily recognized under the microscope by their long branch-like tubular structures called hyphae. Fungi causing ringworm infections produce septate (segmented) hyphae. Some show the presence of spores formed directly from the hyphae (arthroconidia).
Under the microscope Tinea versicolor is recognized by curved hyphae and round yeast forms that give it a spaghetti-and-meatball appearance. Yeast cells appear round or oval and budding forms may be seen. The KOH prep cannot identify the specific organism the specimen can be submitted for fungal culture to identify the organism.
8. A normal, or negative, KOH test shows no fungi (no dermatophytes or yeast). Dermatophytes or yeast seen on a KOH test indicate the person has a fungal infection. Follow-up tests are usually unnecessary.
9. The skin may be sore after the test because of the tissue being scraped off the top of the surface of the skin.
Specimens should be collected in sterile containers or with sterile swabs and transported immediately to the laboratory. This product is used in conjunction with other biochemical and serological tests to identify cultures of isolated organisms.
Mix the specimen with a small drop of India on a clean glass slide. Place a cover slip over the smear and press gently. The preparation should be brownish, not black. Using reduced examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.
Note – The India is ready to use. Further dilution with water is not recommended.
Note – Production of capsular material may be increased by cultivation in a 1% peptone solution (Peptone Broth ).
Fungal cultures are a test to try and ascertain the type of fungal organism that is responsible for an infection or even the presence of the organism in the first place. Fungi are one of the five classes of pathogens that cause disease the other being viruses, bacteria, prions, and helminthes.
It is very important that the correct organism is identified during an infection because the drug treatments differ according to the type of organism and one treatment would not work for the other.
A fungus culture is usually acquired from wound exudates and swabs of areas that are within the body like the mouth and vagina. Very rarely can fungi cause infections within the body because the human immune system is more than adapt at eliminating fungi.
However, sometimes, a fungal blood culture might need to be done because of a condition called fungal sepsis. Sepsis is the presence of bacteria or an infection in the blood. This is a very rare condition and a medical emergency as well. The fungal culture test that is performed in this condition will usually turn up the Candida fungus as the causative organism, though the list is quite endless.
A fungal culture is taken from a part of the body in secretions or by taking a blood sample. Most infections with fungi are limited to the skin, oral mucosa, or the genitals. Very rarely does fungal sepsis actually occur unless a patient is severely immune-compromised. This is usually the case in patients with AIDS or with diabetes. Both of these diseases will cause the immune system to stop working altogether giving rise to all sorts of opportunistic infections.
A fungal growth is acquired by analyzing the secretions and exudates from a wound or by swabbing a surface that is infected. A blood sample might also be required for checking for the presence of fungi in the blood. The sample is then cultured in a fungus-friendly environment for up to a week or more before the fungal colonies become visible.
This stain is prepared over two days.
1. On the first day, dissolve the Cotton Blue in the distilled water. Leave overnight to eliminate insoluble dye.
2. On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker, place on magnetic stirrer until the phenol is dissolved.
4. Filter the Cotton Blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at room temperature.
1. Calcofluor White with 10% KOH
2. Potassium Hydroxide (KOH) with Chlorazol Black
5. Cello tape Flag Preparations
6. Slide Culture Preparations
7. Southgate’s Mucicarmine stain
8. Periodic acid-Schiff (PAS) and PAS Digest stain
9. Grocott’s Methenamine Silver (GMS) stain
Cryptococcus neoformans, because of its large polysaccharide capsule, can be visualized by the India stain. Organisms that possess a polysaccharide capsule exhibit a halo around the cell against the black background created by the India.
Recent studies have detected phylogenetic signals in pathogen–host networks for both soil-borne and leaf-infecting fungi, suggesting that pathogenic fungi may track or coevolve with their preferred hosts. However, a phylogenetically concordant relationship between multiple hosts and multiple fungi in has rarely been investigated. Using next-generation high-throughput DNA sequencing techniques, we analyzed fungal taxa associated with diseased leaves, rotten seeds, and infected seedlings of subtropical trees. We compared the topologies of the phylogenetic trees of the soil and foliar fungi based on the internal transcribed spacer (ITS) region with the phylogeny of host tree species based on matK, rbcL, atpB, and 5.8S genes. We identified 37 foliar and 103 soil pathogenic fungi belonging to the Ascomycota and Basidiomycota phyla and detected significantly nonrandom host–fungus combinations, which clustered on both the fungus phylogeny and the host phylogeny. The explicit evidence of congruent phylogenies between tree hosts and their potential fungal pathogens suggests either diffuse coevolution among the plant–fungal interaction networks or that the distribution of fungal species tracked spatially associated hosts with phylogenetically conserved traits and habitat preferences. Phylogenetic conservatism in plant–fungal interactions within a local community promotes host and parasite specificity, which is integral to the important role of fungi in promoting species coexistence and maintaining biodiversity of forest communities.
Microorganisms and Plant Growth
Rhizosphere refers to the region in the vicinity of roots in which the maximum microbial growth and activities operate (Fig. 34.1). The term ‘rhizosphere’ was first used by Lorenz. Hiltner a German scientist, in 1904.
Greater microbial growth and activities take place in rhizosphere because the roots release a wide variety of materials including various alcohols, sugars, ethylene, amino and organic acids, vitamins, nucleotides, polysaccharides, and enzymes that create unique environments for the soil microorganisms.
The intensity of the microbial growth and activities depends on the distance to which the root exudates can migrate. The rhizosphere microorganisms not only increase their numbers when the root exudates become available, but their composition and function also change.
Rhizosphere microorganisms, as they respond to root exudates, also serve as labile sources of nutrients for other organisms, thus creating a soil microbial loop in addition to playing critical roles in organic matter synthesis and degradation.
Rhizoplane refers to the ‘root surface’ together with closely adhering soil particles. In sampling the system for rhizoplane studies, soil adhering to roots is removed and roots subjected to serial washing by sterilized water (10-12 times) until the clean root surface is exposed.
When such washed roots are plated, characteristic fungi and bacteria appear on agar plates, thereby indicating that there are certain microorganisms intimately associated with the root surface.
Some fungi inhabit the root surface in a mycelial state. They belong to the genera Mortierella, Cephalosporium, Trichoderma, Penicillium, Gliocladium, Gliomastix, Fusarium, Cylindrocarpon, Botrytis, Coniothyrium, Mucor, Phoma, Pythium, and Aspergillus.
Fine structure studies on the epithelial layer of plant roots after inoculation with specific bacteria have shown that bacteria get embedded on the surface of the root with the help of the mucilagenous external layer on the ‘mucigel’ normally present on actively growing root system.
Rhizosphere effect is the direct influence exerted by plant roots on the microorganisms within the rhizosphere. Likewise, the microbial populations in the rhizosphere considerably effect the growth of the plant. As a result of these interactions there is qualitative effect and microbial populations reach much higher densities in the rhizosphere than in the non-rhizosphere soil.
It is now clearly established that greater number of bacteria fungi and actinomycetes are present in the rhizosphere soil than in non-rhizosphere soil and there are innumerable reports in literature to substantiate this fact (Table 34.1). Several factors such as soil type, its moisture, pH and temperature, and the age and condition of plants are known to influence the rhizosphere effect.
Apart from the numerical preponderance of microorganisms in the rhizosphere, the rhizosphere effect is also manifested in the occurrence and distribution of bacteria characterized by specific requirements (Table 34.2) of amino acids, B- vitamins, and specialized growth factors (nutritional groups).
However, greater rhizosphere effect is seen With bacteria than with actinomycetes or with fungi. It is almost negligible with regard to algae and protozoa.
Root Exudates and their Influence on Rhizosphere Microorganisms:
Variety of Root Exudates:
A variety of organic substances available at the root region by way of exudates from roots are one of the most important factors responsible for rhizosphere effect. These substances directly or indirectly influence the quality and quantity of microorganisms in the rhizosphere. The substances exuded by plant roots include amino acids, sugars, organic acids, vitamins, nucleotides, and many other unidentified substances.
The nature of substances exuded by roots of plants has been summarized in Table 34.3. The nature and amount of substances exuded are dependent on the species of the plant, age, and environmental conditions under which they grow.
By the use of 14 CO2, it has been shown that products of photosynthesis are translocated to the root system and find their way into the rhizosphere in less than 12 hours, clearly indicating the influence of the metabolism of plants in determining the extent of the rhizosphere effect.
The root cap and active growth areas are primary regions of root exudation and one of major sites of carbon release from seminal wheat roots into the soil happens to be the zone of the root elongation. It has been advocated that exudation is either from root tips or regions at which lateral roots emerge from the main root.
Following are the important influences exerted by root exudates on soil micro-flora:
1. Root exudates influence the survival and proliferation of dormant structures of various soil surviving pathogens. Spores or sclerotia of many pathogenic fungi such as Rhizoctonia, Fusarium, Selerotium, Aphanomyces, Pythium, Colletotriclium, Verticillium, Phytophthora, and Plasmodiophora have been shown to germinate by the stimulus provided by the root exudates of susceptible cultivars of the host plants. The stimulus for germination has been attributed to compounds exuded by plant roots which help to overcome, in some manner, the static nature of dormant reproductive structures in soil.
2. Root exudates may provide a food base to result in the growth of antagonists which could suppress the growth of pathogenic microorganisms in soil. Many instances have been reported where the rhizosphere of resistant varieties harboured more numbers of Streptomyces and Trichoderma than that of the susceptible varieties.
3. One of the attributes of root exudates is the possible role they play in neutralizing the soil pH and altering the microclimate of rhizosphere through liberation of water and carbon dioxide. Such changes may influence infections of roots by pathogenic fungi.
Changes in Rhizosphere Micro-flora:
Changes in the rhizosphere micro-flora are reported to take place by:
(2) Foliar application of nutrients, and
(3) Artificial inoculation of seed or soil with preparations containing live microorganisms, especially bacteria (bacterization).
Microbial seed inoculants such as Azotobacter, Beijerinckia, Rhizobium, or P-solubilizing microorganisms may help in the establishment of beneficial microorganisms in the rhizosphere or in the immediate vicinity of growing roots.
Field experiments have shown that counts of Azotobacter in wheat rhizosphere increased upon artificial seed inoculation indicating the efficiency of bacterization as a means of altering and improving the rhizosphere micro-flora.
Many experiments have been done to find out the effects of N, P, and K additions on rhizosphere micro-flora. Also, extensive studies have been done on induced changes in the rhizosphere micro-flora by foliar sprays of antibiotics, growth regulators, pesticides, and inorganic nutrients in the hope that such an approach may serve as a new tool in biological control of root diseases. However, no definite conclusions or guidelines have emerged from such studies to merit their application under field conditions.
Nitrogen Fixation by Free Living Microorganisms in Rhizosphere:
Many free living microorganisms in the rhizosphere are found to fix atmospheric nitrogen. These microorganism are Azotobacter, Azospirillum, Klebsiella, Bacillus, Beijerinckia, Pseudomonas, Clostridium, Enterobacter, Erwinia, Derxia, Frankia, etc.
Colonization by Azotobacter has been observed to be limited in the rhizosphere and practibly negligible on rhizosphere. Colonization of rhizoplane by Azospirillum has been noticed with rather extensive intrusion within root tissues. Sporangia of Frankia have been observed in the rhizosphere of Casuarina seedlings.
Associative and Antagonistic Activities:
Many microorganisms depend on in rhizosphere for extracellular products, mainly amino acids and growth promoting factors. This represents their associate functioning in the soil. Co-inoculation of nitrogen fixing Azotobacter and Azospirillum isolates with Rhizobium appears to have beneficial influence in increasing, nodule number, nitrogen fixation, and yield of soybean, pea, and clover.
Russian workers have demonstrated an increase in amino acid content in plants grown in soil inoculated with specific microorganisms. Secretion of antibiotics by microorganisms and the resultant biological inhibition of growth of other susceptible microorganisms are demonstrable in soil as well as in pure cultures.
Such antagonistic effects are natural to expect even in uncultivated soil and from the agronomic point of view excessive inhibition of Azotobacter or Rhizobium in the root region may lead to decreased nitrogen fixation or nodulation.
Plant Growth Promoting Rhizobacteria (PGPR) in Rhizosphere:
The term ‘rhizobacteria’ refers to those non-symbiotic (free living) bacteria which colonize the rhizosphere very aggressively. These free-living rhizobacteria affect the plant growth favourably and, in broad sense, are called plant growth promoting rhizobacteria (PGPR).
PGPR have been discovered by Kloepper et at. in 1980 and belong to genera Pseudomonas, Bacillus, Serratia, Arthrobacter, and Streptomyces. Pseudomonas spp. exhibit fluorescence under ultra-violet light and hence are also known as fluorescent pseudomonads.
Two possible mechanisms have been suggested to explain the beneficial effects of PGPRs in enhancing production.
(i) Competition for substrate and niche exclusion, and
(ii) Production of siderophores and antibiotics. However, more than one mechanism may operate for mediating a biological control.
Fluorescent pseudomonads ‘mop up’ nutrients in the rhizoshpere because of their versatility in growth and nutrient absorption. The points of emergence of lateral roots are favourite spots where DRBs and PGPRs appear to complete for these spots very effectively.
PGPRs are being commercially produced and marketed. A product by name QUANTUM 4000 is being marketed by Gustafson Inc. Dallas, Texas as a growth promoter on peanut (groundnut) and cotton. The product contains Bacillus subtilis strain GBO3, which is a derivative of strain A13.
Other products may follow once procedures are standardized for mass multiplication together with strategies for carriers and quality control. Prior to this the repeatability of success under field conditions have to be established with regard to fluorescent pseudomonads quite often, strains that succeed under green house conditions fail to do so in the field.
The use of multiple strains of fluorescent pseudomonads has shown success in the control of diseases such as take all of wheat and Fusarium wilt of radish.
Encouraging results in the control of take-all diseases of wheat have come in northwest China from field trails covering 4000 ha during 1991-94 by using P. fluoresceins CN12 and Tn5 derivatives, in different sites exhibiting varying environmental conditions the yield increases of wheat due to rhizobacterial inoculation varied from about 16 to 65 percent.
Phyllosphere and Phylloplane:
Phyllosphere refers to the zone on leaves inhabited by microorganisms and the phylloplane represents the leaf surface. The term ‘phyllosphere’ was coined by Ruinen, a Dutch microbiologist, from her observations on Indonesian forest vegetation, where thick microbial epiphytic associations exist on leaves.
Plant parts, especially leaves are exposed to dust and air currents resulting in the establishment of a typical flora on their surface aided by the cuticle, waxes and appendages, which help in the anchorage of microorganisms. These microorganisms may die, survive or proliferate on leaves depending on the extent of influence of the materials in leaf diffusates or exudates.
Leaf diffusates or leachates have been analysed for their chemical constituents. The principal nutritive factors are amino acids, glucose, fructose, and sucrose. If the catchment areas on leaves or leaf sheaths arc significantly substantial, such specialized habitats may provide niches for nitrogen fixation and secretion of substances capable of promoting the growth of plants.
Under conditions of high humidity, as in wet forests in tropical and temperate zones, the microflora in phyllosphere may be quite high.
The dominant and useful bacteria that have been identified are Azotobacter, Beijerinckia, Pseudomonas, Pseudobacterium, Phytomonas, Erwinia, Sarcina. Nitrogen fixing cyanobacteria (e.g., Anabaena, Calothrix, Nostoc, Scytonerna, Tolypothrix) have been encountered in phyllosphere in various moss forests.
Some of the fungi and actinomycetes recorded in phyllosphere are Podospora, Uncinula, Sporobolomyces, Cryptococcus, Rliodotorula, Cladosporium, Altemaria, Cercospora, Helmintliosporium, Erysiphe, Sphaerotheca, Torula, Torulopsis, Oidium, Rhytichosporium, Spermospora, Aureobasidium, Metarrhizium, Myrothecium, Verticilliuni, Melanospora, Saccliaromyces, Candida, Tilletia, Tilletiopsis, Penicillium, Cephalosporium, Fusarium, Periconia, Pithomyces, Mucor, Cunninghamella, Fusarium, Trichoderma, Heterosporium, Stachybotrys, Aspergillus, Curvularia, Rhizopus, Syncephalastrum, Actinomyces, and Streptomyces.
Many bacteria in phyllosphere are considered to fix nitrogen, while the leaves in turn provide carbohydrates and other nutrients to them. Bacteria of the genera Bacillus, Achromobacter, Pseudomonas, Cellulomonas have been isolated from the phyllosphere of pea and wheat and have been proved to be potential nitrogen fixers (Table 34.4).
Phyllosphere microorganisms are thought to effectively control air-borne pathogens from disease causation. These microbes or their propagates induce plants to synthesize ‘phytoalexins’ as defence weapon to counter disease causing pathogens.
Notwithstanding the above observations, the exact role of phyllosphere microorganisms still remains conjectural. Experiment done under laboratory conditions to demonstrate nitrogen fixation in the phyllosphere of several plants by the use of 15 N and the quantitative data obtained arc so divergent that one is led to believe that fixation of nitrogen is a very variable phenomenon in the phyllosphere.
Therefore, experiments have to be designed to study the fate of biologically fixed nitrogen in the phyllosphere. In recent part, spraying of leaves of crop plants with aqueous solutions of sucrose or with bacterial suspensions has resulted in enhanced growth and yield of certain legumes and cereals in pot during experimental trials.
Apparently, such sprays sometimes have intensified the biochemical events on the phyllosphere towards the beneficial side. These observations require to be necessarily evaluated under field conditions so as to exploit the phyllosphere phenomenon towards improvement of agricultural output.
3. Mycorrhizae (Sing. Mycorrhiza):
Mycorrhizae represent a mutualistic symbiosis between the root system of higher plants and fungal hyphae and contribute variously in plant growth promotion.
4. Cyanobacteria (Oxygenic Phototrophs):
The cyanobacteria or the blue green bacteria are amongst the oldest organisms evolved on earth. They represent the only group of oxygen-evolving photosynthetic prokaryotes which possess ability to fix atmospheric nitrogen and promote plant growth.
Frank (1889) first reported their ability of nitrogen-fixation. However, most of the cyanobacteria produce heterocysts that act as the site of nitrogen-fixation.
5. Phosphate Solubilizing Microorganisms (PSM):
Phosphorus occurs in soil in two forms, organic phosphates and inorganic phosphates but deficiency of phosphorus may take place in crop plants growing in soils containing adequate phosphates. This may be partly due to the fact that plants are able to absorb phosphorus only in its available form.
Phosphates present in soil are made available to plants mainly by the activities of some soil microorganisms, which are called phosphate solubilizing microorganisms (PSM).
The latter not only play important role in reducing phosphorus deficiency in plants by way of altering unavailable phosphate into available form, but also they release soluble inorganic phosphate (H7SO4) into soil through decomposition of phosphate-rich organic compounds.
In addition, certain soil inhabiting microorganisms, through assimilation, may immobilize available phosphates in their cellular material and such immobilization processes in soil may contribute to phosphorus deficiency of crop plants.
Although bacteria are used in the large scale preparations of phosphate solubilizing cultures to promote plant growth, fungi such as Aspergillus, Penicillium, Cladosporium, Paecilomyces, Fusarium, Rhizoctonia, etc. appear more advantageous agents in the solubilisation of phosphates. Some fungi (e.g. Glomus, etc.) form associations with plant roots. These associations are called mycorrhizae.
Mycorrhizal fungi convert non- available phosphorus into an available form, produce growth promoting substances, and also protect plants against soil pathogens. Phosphate solubilizing microorganisms usually reduce the pH of the substrate by secretion of various organic acids such as formic acid, acetic acid, propionic acid, lactic acid, organic acid, fumaric acid, and succinic acid.
Phosphate Solubilisation in Different Soils:
Phosphate solubilisation in neutral or alkaline soil is apparently not rare since one-tenth to one-half of the bacterial isolates (e.g. isolates of Pseudomonas, Mycobacterium, Bacillus, Micrococcus, Flavobacterium, etc.) tested usually are capable of solubilizing calcium phosphates, and counts of bacteria solubilizing insoluble phosphates may range from 10 5 to 10 7 per gram. Such bacteria arc often especially abundant on rhizoplanes (root surfaces).
On the contrary, acid soils are generally poor in calcium ions and, therefore, phosphates are precipitated in the form of ferric or aluminium compounds which are not so easily amenable to solubilisation by soil micro-organisms.
In acid soils, however, the deficiency of phosphorus may occur due to the earlier mentioned reason, and this deficiency can be overcome by inoculating seed or soil with phosphate solubilizing microorganisms along with phosphatic fertilizers.
Some bacteria produce iron chelating substances, called siderophores. The siderophores transport iron into bacterial cells. Florescent pseudomonads produce yellow-green, fluorescent siderophores which specifically recognize and sequester the limited supply of iron in the rhizosphere and thereby reduce the availability of this trace element for the growth of the pathogenic micro-organisms. The availability of iron in soil decreases with increase in pH and therefore PSM function better in neutral and alkaline soils than in acid soils.
Ferric Phosphate Mobilization:
Although solubilisation of phosphate usually requires acid production as stated earlier other mechanisms may account for ferric phosphate mobilization. In flooded soil, the iron in the form of insoluble ferric phosphate is reduced resulting in the formation of soluble iron with a concomitant release of phosphorus into solution.
Such increases in the availability of phosphorus on Hooding may explain why rice cultivated under water often requires less amount of fertilizer phosphorus than the same crop grown in dry-lands. Phosphorus may also be made more available for plant uptake by certain bacteria that release hydrogen sulphide (H2S). a product that reacts with ferric phosphate to yield ferrous sulphide, liberating the phosphorus.
Commercialization of PSM:
Phosphate solubilizing microorganisms (PSM) have been found more beneficial in case of vegetable than cereal crops. A commercial preparation, namely, phosphobacterin was widely used for the first time in USSR. This preparation contained bacterial cells of Bacillus megatherium. But, the usefulness of phosphobacterin in soil was rather overemphasized despite the fact that increase in grain yield was of the order of 5-10%.
However, various field trials have been conducted by Indian Agricultural Research Institute (IARI) with wheat, maize, arhar, rice, potato, groundnut, gram. etc. to test the efficiency of phosphate solubilizing bacteria on the crop yield. It was found that significant increases where limited to 13 out of 38 experiments. These results demonstrated that there was no consistent response with respect to increase in yield.
Structure and Characteristics:
Azotobacter is a soil-inhabiting bacterium and comprises large, gram-negative, obligately aerobic rods (Fig. 34.2A). This bacterium freely lives in soil and fixes atmospheric nitrogen nonsymbiotically. The first species of Azotobacter was discovered by the Dutch microbiologist M. Beijerinck in the beginning of 20th century, and was named by him Azotobacter chroococcum.
Subsequently, many other species of Azotobacter were isolated from different soils of the world and some important ones are: A. agilis, A. vinelandii, A. beinjerinckii, A. insignis, A. macrocytogenes, A. paspali, etc. Azotobacter cells are large, many isolates being almost the size of yeasts, with diameters of 2-4 μm or more. Pleomorphism is common and a variety of cell shapes and sizes have been described. Some strains possess peritrichous flagella.
Although the Azotobacter is an obligate aerobe, its enzyme callcd nitrogenasc that catalyzes atmospheric nitrogen fixation is oxygen-sensitive. It has been studied that the high respiratory rate characteristic of Azotobacter and the abundant capsular slime help protect nitrogenase from oxygen.
This bacterium grows on a wide variety of carbohydrates, alcohols, organic acids, amonia, urea, and nitrate. Azotobacter forms cysts (Fig. 34.2B) the resting structures, which arc resistant to desiccation, mechanical disintegration, and ultraviolet and ionizing radiation. Each cyst measures about 3 μm in diameter.
Mechanism of N2 Fixation:
Azotobacter (A. chroococcum and A. vinelandii) is one of the most extensively investigated member amongst free-living nitrogen fixing bacteria. The use of 15 N tracer and acetylene reduction method have however enriched our knowledge regarding the biochemical pathway between atmospheric nitrogen (dinitrogen N2) and ammonia (NH3) but the exact nature of intermediate products have eluded even critical investigators.
Nevertheless, the overall reaction in the enzymic reaction of N2 to NH3 can be postulated as under:
Nitrogenase of Azotobacter:
Nitrogenase, the enzyme that catalyzes atmospheric nitrogen fixation, consists of two protein fractions:
(i) The Mo-Fe containing protein (molecular weight 220,000-2,70,000) and
(ii) Fe containing protein (molecular weight 55,000-66,800).
In Azotobacter (A. vinelandii), two additional nitrogenases have been investigated. One of these possesses vanadium (V) instead of molybdenum (Mo) and the other has neither molybdenum nor vanadium.
The characterization of these nitrogenases has generated fresh problems in pinpointing evidences to demonstrate the essentiality of molybdenum for N2-fixation and characterization of the site at which nitrogen binds to nitrogenase.
Effect of Azotobacter Inoculation:
Azotobacter inoculated seed or soil effectively increase crop yield if the soil is well-manured and contains high organic matter. In addition to fix atmospheric nitrogen, Azotobacter synthesizes substances like B-vitamins. indole acetic acid and gibberellins in pure culture during experimental trails.
Also, the bacterium possesses fungistatic properties to some pathogenic fungi such as Altemaria and Fusarium. These properties of Azotobacter, however, favour the fact that these bacteria promote seed germination and plant growth.
Generally low population of Azotobacter is found in the rhizosphere of crop plants and in uncultivated soil. Often inoculation of soil or seed docs not improve the situation. To overcome this limitation, repeated application of Azotobacter during different stages of growth of a crop is now being recommended with the object of increasing the number of bacteria in soil.
Some experiments on inoculation of soil with Azotobacter with different doses of inorganic N fertilizer have revealed the possibility of saving considerable amount of N fertilizer while still attaining desired yields of rice. Field trials with new and efficient cultures of Azotobacter have shown that the yields of some important crop plants can be substantially increased by Azotobacter inoculation (Table 34.5).
Azospirillum is a rod to spirillum-shaped nitrogen fixing bacterium and freely lives in soil forming nonspecific symbiotic associations with various plants (Fig. 34.3), in particular, corn. This genus consists of species, namely, A. lipoferum, A. brasilense, A. amazonense, A. halopraeferans, A. nitrocaptans, and A. seropedica. Azospirillum lipoferum was originally described and named Spirillum lipoferum by Beijerinck in 1922.
Although this species of the genus was known since 1963 as a nitrogen fixer, it was Dobereiner and colleagues in Brazil who in 1975 highlighted and attributed the nitrogen fixation potential of some tropical forage grasses (e.g., Digitaria, Panicum, maize, sorghum, wheat and rye) to the activity of S. lipoferum in their roots.
Azospirillum characteristically develops white, dense, and undulating pellicles on a semi-solid malate containing enrichment medium. The pellicle is formed 2 mm below the surface of the medium indicating the microaerophilic nature of the bacterium. Azospirillum in gram-negative contains poly-β-hydroxy butyrate (PHB) granules, and shows polymorphism and spirillar movement.
It fixes atmospheric nitrogen (dinitrogen) in microaerophilic surroundings (low oxygen conditions) but possesses ability to grow profusely in ammonium- rich environment without fixing nitrogen. The bacterium also produces growth substances such as indole acetic acid (IAA), kinetins, and gibberellins. Table 34.6 shows the species of Azospirillum and their association partners investigated since 1974.
Effect of Azospirillum Inoculation:
Indian Agricultural Research Institute carried out field experiments to know that effect of Azospirillum inoculation in different parts of India.
These experiments revealed that seed inoculation of sorghum (Sorghum bicolor), bajra (Pennisetum americanum) and ragi (Eleusine corocana) increased grain and fodder yields in different agro-climatic conditions of India (Table 34.7). Similar results have been obtained by scientists in Israel who also find responses of millets to Azospirtilum inoculation.
M.W. Beijerinck, a Dutch microbiologist, was the first to isolate and cultivate a microorganism from the nodules of legumes in 1888. He named it Bacillus radicicola which ensured its place in Bergey’s Manual of Determinative Bacteriology under the genus Rhizobium.
Rhizobia represent the most well known group of symbiotic nitrogen fixers and all rhizobia were previously included in the genus Rhizobium, but later this genus has been split into six genera whose taxonomic position is shown in Table 34.8.
Each of the six genera of rhizobia consists of many recognized species the important ones of which are shown in Table 34.9.
Structure and Characteristics:
Rhizobia are soil inhabiting, free-living heterotropic bacteria which show locomotion with the help of peritrichous or sub-polar flagella. The different strains of rhizobia are attracted by different flavonoids, chemical substance secreted by hosts, and reach the host’s root zone to form symbiotic association to fix atmospheric nitrogen. However, the structure and characteristics of different genera of rhizobia are the following.
Rhizobium is a rod-shaped, 0.5-0.9 x 1.2-3.0 μm long, motile, gram-negative, non-spore forming bacterium. It utilizes organic acids salts as carbon sources without gas formation and grows optimum at 27°C temperature and 6.8 pH. This bacterium for root nodules with its leguminous hosts and fixes atmospheric nitrogen.
The colonics of Rhizobium appear as circular, convex, semitranslucent, raised, mucilaginous, and usually 2-4 mm in diameter. The strains of Rhizobium are fast-growing, where generation time lasts about 6 hours besides showing some other differences with rest of the members of family Rhizobiaceae.
Sinorhizobium, like Rhizobium, is fast growing. It is rod-shaped, usually contains poly-β-hydroxybutyric acid (PBHA) granules, non-spore forming, gram-negative, motile, and aerobic. Most of its strains grow at 35°C temperature and 6.8 pH. Sinorhizobium is a new genus.
It has been observed recently that some rhizobial strains, which are fast growers, nodulate soybean (generally, slow growing bradyrhizobia nodulate soybean). These fast growers were identified as a seperate genus Sinorhizobium.
Bradyrhizobium strains are slow growers with a generation time usually about 12 hours or even more. They move in the soil with the help of one polar or subpolar flagcllum. The growth on carbohydrate medium is accompanied by exopolysaccharide (EPS) slime.
Some strains can grow chemolithotrophically (inorganic salt users) in the presence of H2, CO2, and low level of oxygen. The bacteroids in root nodules are slightly swollen rods with rare branching or coccus forms. The main symbiotic partner of Bradyrhizobium is soybean, while other plants (e.g. Lotus, Vigna, Lupinus, Cicer, Mimosa, Lablab, Acacia, Dalbergia) also are the symbiotic partner.
Mesorhizobium is raised as a new genus of the family Rhizobiaceae only recently and has been named on the basis of whole sequence studies of 165 rRNA. Some of the species of Rhizobium, namely, R. loti, R. haukuii, R. ciceri, R. mediterraneum, R. tianslianense arc now known as the species of genus Mesorhizobium.
Azorhizobium strains bear flagella and are motile. They bear peritrichous flagella on solid medium but one lateral flagellum in liquid medium. They are oxidase and catalase positive and cannot oxidize mannitol. However, Azorhizobium, in contrast to other rhizobia, is a stem-nodulating bacterium.
A caulinodans develops nodules on the stem of tropical aquatic legume Sesbania and fixes atmospheric nitrogen. Stem-nodulated leguminous plants are quite widespread in tropical regions where soils are often nitrogen-deficient because of leaching and intense biological activity.
Root Nodule Formation and N2-Fixation:
The rhizobia grow free-living in soil, infect leguminous plants, and establish a symbiotic existence. Infection of the roots of a legume with the appropriate species of one of five genera of rhizobia leads to the formation of root nodules that fix atmospheric nitrogen. Nitrogen fixation by these symbionts are of considerable agricultural significance as it leads to considerable increases in combined nitrogen in the soil. Since nitrogen deficiencies often occur in unfertilized bare soils, nodulated legumes can grow well in areas where other plants cannot.
9. Actinorhizae (Frankia-Induced Nodulation):
Apart from legumes nodulated by rhizobia, roots of the some non-leguminous plants are nodulated by an actinomycete named Frankia. These actinomycete associations with plant roots are called actinorhizae (sing, actinorhiza). Actinorhizae fix considerable amounts of nitrogen and are important, particularly in trees and shrubs.
There are 25 genera from 8 angiosperm families which have been described to possess actinorhizal root nodules. These families with genera mentioned in parentheses are Casurinaceae (Casurina, Allocasuarina, Centhostoma, Gymnostoma), Coriariaceae (Coriaria), Dasticaceae (Datisca), Betulaceae (Alnus), Myricaeeae (Comptonia, Myrica), Elaeagnaceae (Elaeagnus, Hippophae, Shepherdia), Rhamnaceae (Adolphia, Ceanothus, Colletia, Discaria, Kentrothamnus, Retanilla, Talguenea, Trevoa) and Rosaceae (Cercocarpus, Chaemabatia, Cowania, Dryas, Purshia).
The genera Casuarina for tropical and sub-tropical regions and Ainus (A. rubra. A. glutinosa, A. crispa, A. jorullensis, A. acuminata) for temperate regions stand out as excellent examples for the benefits they provide to the ecosystems by way of nitrogen inputs.
They can adapt themselves to grow under most diverse environmental conditions and geographical zones. Casuarina species (C. equisetifolia, C. cunninghamiana, C. littoralis, C. stricta, C. junghuniana, C. glauca and C. torulosa) provide substantial fuel and building materials in tropical countries while alders provides the most utilised hard wood as well as bark for paper industries in temperate regions.
Frankia is the actinomycete classified under family Frankiaceae, suborder Frankineae, order Actinomycetales, class Actinobacteria. This genus was named after its discoverer Frank in the 1880s. Frankia is filamentous, strcptomycete-like, possesses multilocular sporangia (Fig. 34.4), and forms clusters of spores when a hypha divides both transversely and longitudinally.
It is microaerophilic, grows slowly, forms non- motile spores, and grows in symbiotic association with the roots of earlier mentioned variety of nonleguminous angiosperms.
Entry of Frankia to the Host Plant:
Frankia cells get embedded in a mucilage layer in the root region or the spores may get attached to root hairs the root hairs get deformed or curled. The actual entry of Frankia into root hairs has not been seen but hyphae are seen as simple or multiple threads often branching inside the deformed hair in a host derived cell wall material that is continuous with the root hair cell wall (encapsulation).
The threads could be seen penetrating the cortex and in some root sections, pre-nodule formation can be seen within 10-14 days.
Sooner or later, lateral roots in the vicinity of the primary nodule primordium appear, their meristems undergo branching and progressively get infected with Frankia resulting in the formation of a typical adult nodular structure referred to as a ‘rhizothamnion’. In a sense, actinorhizal root nodule is essentially a modified lateral root.
Structural Organization of Actinorhizal Nodule:
Actinorhizal nodules of Alnus and Casuarina occur in clusters attaining a diameter of 5 to 6 cm somewhat resembling a tennis ball (Fig. 34.5) often weighing up to 444 kg dry weight of nodules/ha. There are two types of structural organization in actinorhizal root nodules: Alnus type and Casuarina type. Alnus-type of root nodules possess many lenticels on nodules that provide ventilation.
Internally, there is a central vascular bundle surrounded by a cortex in which several pockets of Frankia inhabiting zones can be seen containing vesicles that are the sites of nitrogenase activity. Casuarina-type of root nodules possess suberized cells containing the hyphal endophyte with swollen tips.
The suberized cells in Casuarina type of root nodules are impervious to air and hence provide protection to nitrogenase and to the swollen hyphal tips which are belived to be the sites of nitrogen fixation. However, the structural differences in both types are diagrammatically depicted in Fig. 34.6.
Both the types of actinorhizal root nodules differ from legume root nodules formed by rhizobia. Legume root nodules are characterized by a central uniformly infected bacteroid and leghaeinoglobin containing zone surrounded by a tight inner cortex that limits gas diffusion with the vascular bundles lying outside the inner cortex.
Nitrogen Fixation and Ammonia Assimilation by Frankia:
Nitrogenase activity in actinorhizal nodules is host as well as Frankia dependent espeon the morphological state of Frankia whether in the form of spores or hyphae. Nitrogenase has been detected in vesicles as well as hyphae but abundance of vasicles coincides with high nitrogenase activity.
The vesicles contain thick wall that retard O2 diffusion thus protecting oxygen sensitive nitrogenase. Two hypotheses are given to understand the possible mechanism of ammonia assimilation on lines similar in cyanobacterial heterocysts. One hypothesis assumes that glutamine is produced in vesicles and could be transported to vegetative hyphae through the constricted stem cell of the vesicles.
In the hyphae, glutamine would be converted by the enzyme GOGAT to glutamate with one of the resulting glutamates going back to the vesicles to function as an ammonia acceptor for repeating the reaction.
In the second hypothesis, it is considered that the enzyme GS (glutamine synthetase) is not active in ammonia assimilation in vesicles which leads to accumulation of the fixed product in the hyphae and surroundings where it would be assimilated by the GS-GOGAT system, presumably aided by the high affinity ammonia perm-ease present in nitrogen starved hyphae which helps in mopping up all free ammonia.
Reafforestation with Frankia-Inoculated Trees:
In developing countries, deforestation for fuel has rendered the land barren and continuous deforestation of the same land in overpopulated regions of such countries has resulted in soils which remain deficient in nitrogen, the most important element for the normal growth of plants.
One of the least expensive and non-polluting ways to replenish the lost soil nitrogen is reafforestation by planting self-supporting nitrogen-fixing trees.