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Penicillium bilaiae is a heterotroph and a saprobe of the phylum Ascomycota found in soil. The fungus colonizes and acidifies the rhizosphere, increasing phosphate solubilization by lengthening the roots of crops and by chelating cations naturally bound to phosphate that obstruct nutrient absorption in crops like wheat, maize, and field beans. P. bilaiae also prevents plant root lesioning by nematodes. Notable secondary metabolites include citric acid, oxalic acid, and malic acid. P. bilaiae also produces cryoprotectant peptides that help the organism to survive in cold climates.

History and taxonomy
P. bilaiae was first identified in Kiev, USSR by T.V. Chalabuda in 1949. It is named after the scientist V. Bilai.

In scientific literature, this fungus is most commonly referred to as P. bilaiae (P. bilaii) or P. bilaji.

Habitat and ecology
Like most fungi of the Penicillium genus, P. bilaiae is ubiquitous in soil. This species of fungi is saprophytic, gathering nutrients from dead or decaying organic matter.

Since its discovery, P. bilaiae has been found in Southern Alberta, Canada; Great Britain, Kuwait, Northern New Zealand, Spain, Sweden, and Western Australia.

Gram-negative Betaproteobacteria composes 93% of the bacterial community mounted on P. bilaiae, with 89% of the Betaproteobacteria identified as Burkholderia. Burkholderia attach to the hyphae of P. bilaiae in natural soil environments because Burkholderia growth is promoted in the acidic environment created by P. bilaiae.

Laboratory detection
Monoclonal antibodies can be engineered to bind surface antigens like mannan on P. bilaiae 's cell wall in order for this species to be distinguished from other microorganisms present in soil samples.

Growth and morphology
P. bilaiae is a fungus of the phylum Ascomycota that makes asexual conidia. Conidia, produced externally on conidiophores by phialides, are rough, or rugose-walled, with spherical to ellipsoidal shape. Conidiophores, the stalks on which the mitospores are generated, are smooth-walled and monoverticillate (non-branched). Phialides, located at the tip of conidiophores, are ampulliform (flask-shaped) and made up of dense and parallel columns. Hyphae are aerial, forming a mycelium of white pigmentation, plane or radial topography, and floccose to funiculose texture.

Microcycle conidiation occurs to preserve species survival when the nutrients necessary for energy production are withheld from P. bilaiae. Growth of spores is reduced and spore volume is decreased.

At room temperature, a colony of orange-brown P. bilaiae grows on a plate of Czapek yeast autolysate agar (CZA) at a rate of 25-33 mm. Reverse colour on CYA appears deep brown. At a temperature of 30°C on CYA, the growth rate of P. bilaiae is 25-30 mm. At a temperature above 25°C, P. bilaiae growth is variable but this fungus typically grows at a rate of 13-15 mm on CYA at 37°C, which is significant when trying to differentiate between Penicillium bilaiae and other species in its clade. P. lilacinoechinulatum, for example, shows only 2-4 mm of growth on CYA at 37°C. P. bilaiae colonies on CYA at 37°C appear white, with yellow or brown soluble pigment.

On malt extract agar (MEA), colony diameter at room temperature is anywhere from 18-28 mm after 7 days. Colonies appear plane or slightly radial with velvety and funiculose mycelia. Under the same conditions, 25% glycerol nitrate agar (G25N) fosters colony growth of 13-17 mm.

On a creatine sucrose agar (CREA) at room temperature, P. bilaiae produces a strong acid. Furthermore, in buffered media P. bilaiae maintains the ability to lower pH.

Pitt (1979) records that P. bilaiae is synonymous with P. lilacinoechinulatum but, while P. bilaiae and P. lilacinoechinulatum are members of the same clade, meaning they have evolved from the same common ancestor, their gene sequences show that they are two distinct species and their growth and morphological characteristics on media differ. For example, in comparison with P. lilacinoechinulatum on CYA, the presence of brown exudate and faster growth at 37°C indicates P. bilaiae.

In addition, P. bilaiae can be differentiated in the laboratory from the similar soil species P. chermesinum by its slower colony growth on CYA and MEA as well as by its rugose conidia.

Nematicidal metabolites
P. bilaiae produces four known nemacitidal compounds: penipratynolene, 6-methoxy-carbonylpicolinic acid; 2,6-pyridinedicarboxylic acid, and aspyrone. The former three compounds, however, have much more potent nemacitidal properties when used against the parasitic nematode Pratylenchus penetrans, which lesions the roots of various crops and causes necrosis.

A 300 mg/l sample of P. penetrans, when treated with metabolite penipratynolene, exhibits 77% mortality. Penipratynolene, also known chemically as methy(2'R)-4-(2'-hydroxy-3'-butynoxy)benzoate, has a colourless, needle-like structure. The same concentration of P. penetrans, when treated with 6-methoxy-carbonylpicolinic acid and 2,6-pyridinedicarboxylic acid, exhibits 52% and 98% mortality, respectively. It has been suggested that the nematode specifically responds to the acetylene compounds of penipratynolene and the carboxy groups in 6-methoxy-carbonylpicolinic acid and 2,6-pyridinedicarboxylic acid.

Acidic metabolites
The metabolites secreted by P. bilaiae that significantly decrease the surrounding pH include citric acid, oxalic acid, and malic acid. These organic acids are directly involved in the process of phosphate solubilization. Their production persists in buffered growth media.

The nutritional conditions in which P. bilaiae employs citric acid and oxalic acid to solubilize inorganic calcium phosphate vary. When high levels of oxygen concurrent with low levels of nitrogen are supplied, citric acid production is optimal. Optimal oxalic acid is secreted by P. bilaiae when carbon is a limiting nutrient, and when citric acid is consumed. Optimal acidification is associated with increased phosphate solubilization. Furthermore, the phase of growth of P. bilaiae affects metabolite production. During the characteristic growth stage, citric acid is secreted but not oxalic acid. During the subsequent plateau of the growth phase called the stationary phase, both metabolites are produced. When P. bilaiae sporulation is induced in the course of rhizosphere acidification, the media becomes alkaline rather than acidic, but at a slower rate than acidification normally occurs. The alkalinization is likely due to the differences in metabolism that exist between the sporulating and vegetative hyphal state.

Albeit at much lower concentrations than citric, oxalic, and malic acid, malonic acid, maleic acid, succinic acid, fumaric acid, and trans-aconitic acid are also secondary metabolites of P. bilaiae.

Weakly cytotoxic metabolites
A total of 18 secondary metabolites were identified in an Australian marine-derived P. bilaiae (MST-MF667) when ethyl actetate (EtOAc) was partitioned. Among them, four notable aromatic polyketides were found: non-polar polyketide citromycetin, citromycin, and di-hydro analogues (-)-2,3-dihydrocitromycetin and (-)-2,3-dihydrocitromycin; as well as diketopiperazines (DKPs) named bilains A-C. The citromycetins and the diketopiperazines found in this isolate were reported as “weakly cytotoxic”. Citromycetin and 2,3-dihydrocitromycetin are also the source of yellow pigments.

Antibacterial activity
P. bilaiae strains isolated from the roots of two types of date palm trees, healthy and brittle leaf diseased, in Tunisia were collected to determine their antibacterial capabilities. Agar-well diffusion tests indicate that P. bilaiae produces a significant zone of inhibition against the Gram-positive bacteria Micrococcus luteus. The metabolites directly responsible for this inhibition were unidentified.

Catecholate siderophore
Siderophore production by P. bilaiae is seen on malt extract agar (MEA) supplied with high iron. Pistillarin, a siderophore containing the compound catechol, has been identified as a metabolite of P. bilaiae only twice.

Thermal generalist
Fungi are ectotherms, deriving heat from their environment but varying temperature does not inhibit the growth rate P. bilaiae, which is a trait not observed in all fungi. P. bilaiae is able to grow quickly at soil temperatures ranging from 17°C to 28°C. Specifically, urban soil-borne P. bilaiae grows equally fast or faster than rural soil-borne P. bilaiae at all temperatures measured at 2°C intervals between 17°C and 28°C degrees, but both urban and rural P. bilaiae show a positive correlation between increased temperature and increased growth. The ability of urban P. bilaiae to better adapt to the increase in temperature is likely due to the urban heat island effect. Because the amount of energy produced in the city makes it comparably warmer, usually by 2-5°C more than rural regions, the P. bilaiae isolates living in urban soils will have adapted better to the increased temperature compared to rural isolates. The marginal difference between urban and rural fungal growth in growth at each temperature interval, however, suggest that the P. bilaiae species is a thermal generalist.

Cryoprotectant polypeptides
P. bilaiae (No. 205) found in Antarctic soil produces antifreeze proteins (AFPs), also termed ice crystal structure-controlling materials (ICSCs), against ice crystal formation. P. bilaiae exhibits thermal hysteresis (TH) at less than 1°C above freezing point.

Agricultural use
Phosphate-solubilizing fungi (PSF) like P. bilaiae make naturally inaccessible soil phosphate accessible to the roots of plants. Plants then use the organic phosphate to make cellular macromolecules.

P. bilaiae-inoculated canola and wheat show increased phosphate uptake and increase dry matter yield. The root length of pea plants increased by 48% when treated with P. bilaiae under conditions in which phosphate was a limiting nutrient. The acidification of the microenvironment at the roots of these plants modulates phosphate solubilization. Oxalic acid and citric acid chelate the contaminating cations bound to phosphate in the process.

Soil characteristics like soil pH, texture, and temperature can foster or deter the PSF’s ability to carry out this function. Generally, phosphate solubilization occurs best at high temperatures and in acidic soil. Plant root characteristics such as diameter and length will also affect phosphate solubilization. Thus, incompatibilities may exist between fungi and their environment that will consequently affect their phosphate-solubilizing capabilities.

Phosphate solubilization by P. bilaiae is important because crop yield generally decreases due to phosphate stress particularly in the early season. Supplying crops with living organisms like phosphate-solubilizing fungi instead of chemical biofertilizers is environmentally friendly, safe, and less expensive to implement. It is also reliable as there is no risk of chemical immobilization or degradation in the soil.

Commercial biofertilizer, JumpStart, marketed by Monsanto BioAg, contains the active ingredient Penicillium bilaii, which can be applied to canola, lentil, pea, corn, soybean, oilseed rape, wheat, and barley crops for increased yield. Storage of P. bilaiae as commercial biofertilizer requires convective air drying to preserve the conidia at low water activity levels and slow the metabolism of the fungi. The addition of sugars and polyols protect the fungi’s enzymes and the integrity of their membranes.