TERM PAPER

“MICROBIAL PHYSIOLOGY AND METABOLISM”

Topic: - PAECILOMYCES LILACINUS

D.O.S:-19-11-10

SUBMITTED BY:Mr:- SHASHI SHARMA

Roll no :- B-15 Reg. No:-11006142

Section:-P8003

TABLE OF CONTENT

1. ABSTRACT

2. INTRODUCTION 3. DIVERSITY 4. IMAGES 5. TAXONOMY 6. DESCRIPTION 7. LIFE CYCLE 8. PRODUCT DETAILS 9. SOIL APPLICATION 10. PLANT PARASITIC NEMATODES 11. METHODS FOR NEMATODE CONTROL 12. SOLID STATE FERMENTATION 13. WEST INDIAN JOURNAL 14. BIOCONTROL AGENT 15. METHOD OF APPLICATION 16. FREQUENCY OF APPLICATION 17. DOSAGE 18 .REFERENCES

ABSTRACT

GA Paecilomyces lilacinus is an agent for the potential biological control of soil

nematodes. Arbitrarily primed PCR was used t o fingerprint the genomes of 28 isolates

of this fungus. Most (72%) of the isolates originated from soil of different regions of

Brazil. Fourteen 10-mer oligonucleotide primers of arbitrary sequence revealed 293

scorable binary characters. Distinct genotypes were obtained for each isolate. Cluster

analysis showed a high level of variability among these genotypes. The similarity

among pairwise

comparisons of the isolates varied from 84.3% to F6%, with a mean of 6305%. No

clearly defined phenetic groups were identified by cluster or multivariate analyses. No

correlation with geographical origin or host was detected. In addition, PCR with four

pairs of consensus tRNA gene primers was performed on a subsample of 12 P. lilacinus

isolates, three P. farinosus isolates, two P. fumosomseus isolates, and one isolate of P.

amoenomseus. An inferred phylogeny based on 112 binary characters obtained by

tRNA-PCR showed a monophyletic group which contained most of the P. lilacinus

isolates. In contrast, three isolates of P. farinosus were not in a monophyletic group

under the inferred phylogeny. These results suggest that tRNA fingerprinting could

provide a valuable tool which could be used to develop the molecular taxonomy of

Paecilomyces, as morphological characteristics of asexual structures cannot entirely

resolve species.

Avail from us superior quality Paecilomyces lilacinus which are basically filamentous

fungus, used to kill harmful root-knot nematodes. These are formulated by our experts

as per the varied requirements of the client. These belong to the family of

Trichocomacea.

INTRODUCTION

Paecilomyces lilacinus is effective in plant parasitic nematodes. At GreenMax AgroTech,

Pacilomyces lilacinus is available as talc based formulation with high density spore population. This

product is also available as combined formulation with Beauveria bassiana. Paecilomyes lilacinns is a

typically soil-borne hyphomycete that has been isolated in different parts of the world, especially in

warmer regions (Domsch & Gams, 1980). P. lilacims has remarkable versatility and can survive on

diverse substrates and infect different hosts (Samson, 1974). It can live as a mycoparasite that

colonizes sclerotia of several species of fungi (Gupta e t al., 1993). Also, it has been reported to infect

eggs and cysts of nematodes (Carneiro, 1992). Although the activity of P. lilacintls on insects is

infrequently reported, there is one report of the use of this fungus to control populations of

Nilaparvata ltrgens in rice fields (Rombach e t al., 1986). Recently, an isolate of P. lilacinns has been

studied as a potential biological agent to control eggs of both the nematode Meloidogyne javanica and

the corn root worm, Diabrotica speciosa, in Brazil (Tigano-Milani e t al. , unpublished data). P.

lilacintrs was the most frequently isolated species, when a survey of selective isolation of

entomopathogenic fungi was conducted on soil samples from different regions of Brazil (Chase e t al.,

1986 ; Tigano-Milani e t al., 1993). To study the efficacy of this potential biopesticide, it will be

necessary to characterize the apparently large endemic, soil-borne population. Environmental release

will also require means for positive identification of strains. Genetic markers can be used to study

populations of P. lilacinas. Markers will be required to evaluate the efficacy of treatments, to study

population dynamics and to allow characterization of genetic variation in P. lilacinns. Until now, P.

lilacintrs has been characterized mainly by virulence tests or biochemical characters (Samson, 1974;

Carneiro, 1992). However, it is difficult to associate variability of these phenotypic characteristics

with overallgenetic and phylogenetic relationships. Environmentally sound and economically feasible

alternatives for pest control are nowa subject of numerous studies due to the development of resistance

to pesticides intargeted pathogens, as well as the withdrawal of commercial pesticides from the

marketdue to environmental and public health concern. Plant-parasitic nematodes, especially root-knot

nematodes (RKN), are major pests of several economically important crop plants, causing severe

yieldloss. Methyl bromide, the most widely usedsoil fumigant against nematodes, has beenbanned in

most developed and developing countries, because of the serious threats it poses to the environment.

So there is an increasing demand all over the world forecofriendly nematicides. Biological control

agents (BCAs), such as fungi, offer great scope for field application, but the development of a viable

bioprocess for its commercial production is not an easy task. Paecilomyces lilacinus (Thom) Samson

is a known soil hyphomycete, and it parasitizesRKN eggs and females showing great nematicidal

activity. Growth physiology of filamentous fungi is an important factor considering their production as

BCAs. The German manufacturer Prophyta produces a commercially patented strain of P. lilacinus,

which is under continuousresearch worldwide. Field applications of BCAs are mainly accomplished

by means of fungal conidiospores, which must be virulent and viable for long periods of storage. Solid

state fermentation (SSF) offers many advantages for large scale and cost effective production of

conidiospores. The present review deals with the relevance of controlling RKN in agriculture, and the

application of the BCA P. lilacinus produced under SSF. Existing methods for controlling nematodes

are discussed with emphasis on biological control research and practices using the spores of P.

lilacinus.

DIVERSITY

Paecilomyces lilacinus is a common saprobic, filamentous fungus.It has been isolated from a wide range of habitats including cultivated and uncultivated soils,forests,grassland,deserts, estuarine sediments and sewage sludge. It has also been found in nematode eggs, and occasionally from females of root-knot and cyst nematodes. In addition, it has frequently been detected in the rhizosphere of many crops. The species can grow at a wide range of temperatures – from 8°C to 38°C for a few isolates, with optimal growth in the range 26°C to 30°C. It also has a wide pH tolerance and can grow on a variety of substrates. P. lilacinus has shown promising results for use as a biocontrol agent to control the growth of destructive root-knot nematodes.

Divergent phialides and long, tangled chains of elliptical conidia borne from more complex fruiting structures characteristic of Paecilomyces lilacinus; magnification

460X.

Scientific Classification

Kingdom:Fungi

Phylum: Ascomycota

Class: Ascomycetes

Order: Eurotiales

Family: Trichocomaceae

Genus: Paecilomyces

Species: P. lilacinus

IMAGE OF PAECILOMYCES LILACINUS :-

Paecilomyces lilacinus

Taxonomy

P. lilacinus was classified with the Fungi Imperfecti or Deuteromycetes, fungi for which perfect (i.e.,

sexually reproducing) states have rarely been found. Paecilomyces lilacinus is classified in the section

Isarioidea, for which perfect states have not been found. Many isolates of P. lilacinus have been identified

from around the world and it is accepted that variation exists within the species

Description

P. lilacinus forms a dense mycelium which gives rise to conidiophores .These bear phialides from the ends of which spores are formed in long chains. Spores germinate when suitable moisture and nutrients are available. Colonies on malt agar grow rather fast, attaining a diameter of 5–7 cm within 14 days at 25°C, consisting of a basal felt with a floccose overgrowth of aerial mycelium; at first white, but when sporulating changing to various shades of vinaceous. The reverse side is sometimes uncolored but usually in vinaceous shades. The vegetative hyphae are smooth-walled,hyaline, and 2.5–4.0 µm wide. Conidiophores arising from submerged hyphae, 400–600 µm in length, or arising from aerial hyphae and half as long. Phialides consisting of a swollen basal part, tapering into a thin distinct neck. conidia are in divergent chains, ellipsoid to fusiformin shape, and smooth walled to slightly roughened. Chamydospores are absent.

Life cyclesP. lilacinus is highly adaptable in its life strategy: depending on the availability of nutrients in the surrounding microenvironments it may be entomopathogenic, mycoparasitic, saprophytic, as well as nematophagous.

SCOPE

Brandname: Gmaxbioguard:- :-

ProductDetails:

Technical: Mother culture of Pacilomyces lilacinus was sourced from Project Directorate of Biological

Control (PDBC), Bangalore. Pacilomyces lilacinus, mass multiplied from virulent and pure mother

culture is supplied as talc based formulation. The product will have a minimum population of 1x 107

(CFUs)/g.

Formulation : Talc carrier based product. The product has minimum shelf life of one year from the date of

manufacture. 

Composition:

Pacilomyces lilacinus 1% (w/w) Sticking agent – CMC – 1%.  Inactive Ingredients 98.0% (w/w)

o                   (Moisture 35%, talc 63%)

Packing:-    The product is available in attractive one kg laminated poly pouches. The packing is moisture and

proof and well tolerates transportation and handling. The product is also supplied in bulk packing of

50kg/25 kg sizes in HDPEbags.     

DESCRIPTAION ABOUT THE PRODUCT:- Paecilomyces lilacinus is a common saprophytic, entomopathogenic, mycoparasitic, saprophytic, as

well as nematophagous, filamentous fungus. It is parasitic on nematodes infecting eggs, juveniles,

and adult females of root-knot and cyst nematodes. The species can grow at a wide range of

temperatures – from 8°C to 38°C for a few isolates, with optimal growth in the range 26°C to 30°C. It

also has a wide pH tolerance and can grow on a variety of substrates. P. lilacinus has shown

promising results for use as a biocontrol agent to control the growth of destructive root-knot

nematodes. Paecilomyces protects the roots against plant parasitic nematodes, specifically root-knot

nematodes (Meloidogyne spp.), Banana nematodes (Radopholus similis) reniform nematode

(Rotylenchulus reniformis), and citrus nematodes (Tylenchulus semipenetrans). These nematodes

infect horticultural crops of economic importance. 

PLANT-PARASITIC NEMATODES

Nematodes are roundworms that belong to the phylum Nematoda. They are the

most abundant creatures on earth, occupying different ecological niches and

living as parasites of humans, animals and plants. Parasitic nematodes can cause

a large-scale multiplication and invasion of their host. Phytoparasitic nematodes

can devastate several economically important crops, causing significant losses in

yield. These nematodes are obligate parasites, and they have developed different

parasitic strategies and relationships with their hosts to attain enough nutrients

for development and reproduction. The products of nematode parasitic genes can

be expressed as morphological structures (e.g., stylet), which allow researchers

to assess the level of parasitism in a particular host plant, where nematodes can

develop critical physiologicalfunctions

in the interaction with their host.

METHODS FOR NEMATODECONTROL

Since 1950, the control of phytoparasitic nematodes has been based on chemical

pesticides, although several of them are being withdrawn from the market due to

issues related to the environment lic health. Methyl bromide was widely used

against nematodes, but now it has been withdrawn from the market because of

its adverse effects on the ozone layer. Nematodes also developed resistance

against most of the known pesticides, and this triggered worldwide research for

new alternative agents and methods for nematode control. Possible control

measures change with climate conditions, socio-economical situation of the

country, crop economy, availability of chemical pesticides, resistant cultivars,

and the suitability of agricultural practices.

Resistant plants:-

Plants are resistant to nematodes when they have a reduced level of

reproduction. Nematode resistance genes are present in several crops, and are

an important component of various multiplication programs in tomatoes,

potatoes, cotton, soybean, and cereals. Resistance to nematodes can be either

broad with action against several species of nematodes or narrow against only

selected specific biotypes113. Several resistance genes, dominant or

semidominant, were identified, cloned, and subjected to various studius.

Crop rotation:-

Important method for maintenance and improvement of soil fertility, and for

enhancing yield. In crop rotation, various crops are followed in a certain order in

the same soil. With the same succession of crops reproducing in a regular time

cycle, rotations can be biennial, triennial, and so on. Crop rotation is a very good

strategy that can always be adopted against nematode species with narrow

ranges of plant-host, which is not the case of Meloidogyne sp. However, the order

of plants and the time intervals between susceptible crops depend on the

nematode species.

Chemical control:-

Plant-parasitic nematodes are more vulnerableas juveniles (J2) in soil, when

searching for the roots of host plants. Once an endoparasitic nematode species

penetrates a root, chemical control is more difficult as compounds have to be

non-phytotoxic. There are several nematicides that can be used effectively

against nematode pests of many annual crops, but there appears to be little

progress for management of nematodes in many susceptible perennial crops

without repeated application of nematicides36. There are two kinds of chemical

products that can be utilized against plant parasitic nematodes: soil fumigants

and nematicides. Their application to soil depends on the form of the formulation,

it can be by injection, spraying, mechanical means, or through irrigation pipes

ucts are usually applied before planting and, in the case of pesticides, they are

applied at the time of planting. Fumigants are highly effective against

nematodes, their efficacy is related to their high volatility at ambient

temperatures. All fumigants have low molecular weights, and are available as

gases or liquids. As they volatilize, the gas diffuses through the spaces between

soil particles where the nematodes are killed. The most widely used fumigant is

methyl bromide, which is mainly applied for high valued crops, such as

strawberries and tomatoes, and in lesser amount to grains and commodities.

However, methyl bromide has been banned in developed countries since 2005. In

developing countries, substances with methyl bromide will be withdrawn from

field application by the end of 2015.Other fumigants, such as chloropicrin,

dazomet and meta sodium showed good activity against nematodes when

applied.

.

Biological control :-

An eco-friendly pest management strategy that utilizes deliberate introduction of

living natural enemies to lower the population level of a target pest. These

enemies are commonly referred to as BCAs, which must demonstrate some

characteristics for success in the field, including ability

for rapid colonization of the soil, persistence, virulence, predictable control below

economic threshold, easy production and application, good viability under

storage, low cost of production, compatibility with agrochemicals, and safety. In

nature, it is observed that many natural enemies, such as viruses, bacteria,

rickettsias, fungi, and others, can attack plant parasitic nematodes, but in the

search for suitable BCAs more attention has been given to fungi and bacteria.

Biological control can be either natural (i.e., when a natural population of a

particular organism inhibits the growth and development of nematodes), or

induced (i.e., when BCAs have been introduced artificially). There are two

approaches for introduction: microbial pesticide application for rapid control of a

pest, and the introduction or mass release of a biocontrol agent to provide long

lasting control. The suppression can be specific or non specific, when only one or

two organisms are involved. Researchers have made several attempts to utilize

bacteria for nematode control.

GROWTH PHYSIOLOGY OFFILAMENTOUS FUNGI

Spore production of filamentous fungi is an important stage in its reproduction.

Spore production consists of the formation and liberation of conidiospores. Life-

cycle of imperfect fungi comprises five steps, which are dormancy of the spore,

germination, development of apical mycelium, and conidiogenesis. Normal

development of the mycelium and suitable conidiogenesis are the main

conditions required for a successful sporogenesis. The conidiospore production is

directly related to the quantity and nature of carbon and nitrogen sources

available in a culture media, and it depends on several other factors including

method of inoculation, media salinity, carbon/nitrogen ratio, aeration, water

content, among others85. Conidiospores are characterized by a low water activity,

absence of cytoplasmic movements, and reduced metabolic activity. Under

favourable conditions, spore germination takes place through the formation of a

vegetative tube, which will be the base of a future mycelium. A spore is

considered as germinated when the length of the longest germ tube is greater

than the dimension of the swollen spore. Different techniques, other than

microscopic examinations can be used to assess spore germination. Gompertz

equation and logistic function can be used for analyzing germination data.

Determination of optimal culture conditions for the large-scale production of

conidiospores of filamentous fungi, which are used as BCAs, is highly significant

for commercial applications. There are several studies carried out to enhance

conidiospore production for BCAs.

SOLID STATE FERMENTATION

SSF can be defined as the growth of microorganisms in a moist solid substrate in

the absence of liquid water. The water content in the moist solid substrate must

be adequate to support growth and metabolism of microorganism. SSF can be

carried out in two types of matrices, either in a natural substrate acting as solid

substrate and a source of nutrients or a nutritionally inert support which must be

impregnated with a liquid nutritive media. The most widely used substrates are

of amilaceous or lignocellulosic origin. Several materials are utilized as inert

supports for SSF, such as sugar cane bagasse, amberlite, vermiculite,

polyurethane foam, and polystyrene beads. SSF has several advantages over

SmF, but the choice of the method should depend on the physiology of the

microorganism and the end product. Comparative evaluations of SSF and SmF

indicated several advantages of SSF processes: simplicity of culture media;

absence of liquid residues;

reduction of contamination due to low water content; culture conditions mimic

the natural environment; ease of aeration (humid or dry) because of porosity of

the material; direct utilization of the fermented material; easy downstream

processing because of high yields; and easy to dehydrate and dry the fermented

product in situ. There are certain disadvantages associ ated with SSF processes

which include: excess of heat generation and subsequent difficulties in heat and

mass transfer, problems with the control of fermentation parameters (e.g., pH,

water content), difficulty in biomass estimation and pre-treatment of the

substrate, among others. SSF processes simulate the living conditions of many

higher filamentous fungi. Hence SSF is the cultivation method of choice for

biotechnological processes, where it is required to consider morphological and

metabolic differences in substrate- penetrating and aerial hyphae (e.g.,

production of conidiospores). There is a lack of information about the influence of

physico-chemical and nutritional parameters on the physiology and kinetics of

growth and sporulation of P. lilacinus under SSF, and the methods for estimation

of biomass. As filamentous fungi grow, hyphae penetrate into the solid matrix

becoming impossible to separate substrate from the mycelium, and thus making

difficult a direct measurement of biomass. Indirect methods for estimation of

biomass are available through the analysis of biomass components, such as

glucosamine,nucleic acids, and proteins. Several aspects should be considered

when

selecting biomass components for assay:

1) They should be major components in the microorganism;

2) They should have little or no influence from the substrate; and

3)They must be consistently present throughout development.

The most important method so far to assess biomass consists of measuring the

production of CO2 and the consumption of O2 by the microorganism during

fermentation. This permits the estimation of biomass and specific growth of the

microorganism inside the reactor through correlations between biomass

synthesis and oxygen consumption. .Important factors in SSF Factors affecting

SSF are purely based on the type of microorganism that is employed in the

process. The microorganism in a SSF process can be either natural microbiota of

the substrate or a pure culture. Ensiling and composting are two methods that

utilize the natural microbiota. Pure cultures are mainly used for the production of

fungal conidiospores, secondary metabolites, antibiotics, and other high value

product. There are several groups of microorganisms which can profusely grow in

solid substrates; however, filamentous fungi are well known for their capacity to

grow in substrates of relatively low moisture content due to their physiological,

enzymological, and biochemical properties. The growth of filamentous fungi takes

place combining the apical extension of hyphae and the generation of new

hyphae by mycelial ramification. This allows fungal growth within the solid matrix

to form a solid structure.

The penetration of hyphae into the substrate enhances the access to available

nutrients,

promoting suitable metabolic activity83.In SSF, the quantity of water present in

the media is a function of the substrate water retention capacity. This quantity

should be sufficient for the growth of microorganisms, without destroying the

solid structure or reducing the porosity of substrate or support. The water

content in the substrate influences the morphology of the microorganisms, and

serves as a carrier for enzymes, nutrients and metabolites, as well as in the

solubilization of oxygen. High moisture content of the substrate can lead to

reduced porosity of the solid matrix, weak oxygen diffusion and a high risk for

bacterial contamination. Low moisture levels result in limited growth of the

microorganism, as the distribution of available nutrients in the substrate is not

uniform. The control and monitoring of the gaseous environment in aerobic SSF is

a critical factor for the growth of microorganisms, which depends on the air flow

rate through the substrate and the rate of O2 consumptionAeration provides O2

for aerobic growth and fungal metabolism, and it also helps to control moisture

and temperature, and to eliminate CO2 and some other volatile metabolites. Any

adverse change in the gaseous environment may significantly affect the levels of

production of biomass and enzymes. The initial pH of the substrate in SSF is

usually adjusted to support optimal growth of the microorganism. The pH value

varies according to metabolic activity of the microorganisms. Acid production

during fermentation or the formation of urea tend to decrease the pH. Sudden

and drastic change of the substrate pH can be avoided using a solution of

mineral salts with bufferingcapacity, as suggested by Raimbault and Alazard.

SSF and the production of Biological ControlAgents (BCAs) :-

The most important aspect to be considered when selecting a BCA for

commercial application is the availability of a cost-effective production and

stabilization technology for manufacturing an effective formulation. BCAs are

mainly applied as spores and SSF offers several advantages: 1) The production of

aerial fungal spores are more tolerant to UV radiation; 2) Higher spore stability;

3) Spore resistance to drying; and 4) Higher spore germination rates for longer

storage periods. These better characteristics can be attributed to the presence of

a hydrophobic rodlet layer formed during the production process1. Another

advantage of SSF for production of BCAs is the utilization of agricultural by-

products as substrate for fermentation. The generation of high

amounts of agricultural residues causes serious environmental problems

worldwide, so SSF allows efficient utilization and value addition. The use of

agricultural byproducts for SSF leads to a less expensive process for the

production of BCAs on a large scale. SSF processes are usually cost effective, and

they require reduced labour. In many cases, the fermented substrate can be used

for field application, and thus most technical difficulties associated with

downstream

processing and product formulation aren ruled out. The products of SSF are

usually

air dried or rotavapor dried to be used directly. Roussos et al. studied different

methods for the conservation of fungal spores produced under SSF, and found

that temperature has a significant effect on spore viability in long term storage.

The mass production of fungal spores must be achieved for any commercial

application of BCAs. Therefore, further indepth studies for scaling up SSF

processes are needed, such as the design and development of automated SSF

bioreactors.

Bioreactors employed in SSF processes should provide adequate environmental

conditions for the maximal growth and activity of microorganisms. SSF

bioreactors have been studied for the commercial production of biopesticides,

metabolites, fermented foods, and other product. Several problems in scaling up

SSF processes have been found, such as variations in biomass production, high

inoculum level, substrate sterilization, heat generation due to microbial metabolic

activity, on-line monitoring of aeration, or pH. Bioreactor design for a particular

SSF process should consider: the type of substrate or support to be used, particle

size and mechanical resistance of the substrate, oxygen transfer, nature of the

gaseous phase between the particles of substrate or support, morphology of

microorganisms, and a suitable sterilization process. SSF bioreactors can be

classified in accordance with the quantity of substrate utilized in the process.

They are divided into two categories: laboratory scale (gkg capacity) and pilot or

industrial scale (kg-tons). Another type of classification is based on the design of

fermenters, which may provide agitation or aeration devices. Laboratory scale

bioreactor comprises simple devices, such as petri dishes, Erlenmeyer flasks, jars

and Roux bottles, which are mainly utilized for screening of microorganisms and

substrates. These small bioreactors cannot provide aeration and agitation

controls. Autoclavable plastic bags are also useful and commonly used for the

production of fungal inoculum. The utilization of plastic bags for the production of

fungal spores from Pochonia chlamydosporia has been reported (substrate: rice

and corn grains) for application as a BCA against nematodes111. Column type

bioreactors are well studied as they provide on-line information of the

microorganism’s respiration. These reactors monitor respiration and other

gaseous exchanges, and are mainly used for process optimization studies.

Column bioreactors can also monitor and control aeration, so they are used as a

model for designing and manufacturing several other types of reactors. The

substrate can be cooled by evaporation, and heat generation can be minimized

through convection and heat exchange by glass walls with the help of a water

bath. Reactors designed on continuous agitation are called rotating drum

reactors. These are perforated drums with a horizontal paddle mixer. Rotating

drum reactors were designed to increase contact between the reactor wall and

solid media, as well as to facilitate oxygen transfer. However, they have several

disadvantages, such as agglomeration of substrate, difficulties in temperature

regulation, low oxygen transfer, and alterations of substrate structure due to

intense agitation. Using a similar device from the Zymotis bioreactor for reducing

heat and providing high air flow, a novel bioreactor was designed and patented

by the German company Prophyta. This new bioreactor is exclusively used for the

production of the BCA P. lilacinus, strain PL-251. It has perforated plates where

heat exchangers are located at the bottom, and the substrate on the top. The

flow of sterile air is facilitated by perforated plates. Another reactor patented by

Durand et al was used for the production of fungal conidiospores in biological

control. This reactor has a capacity of 50 L, and it is fitted with a planetary

agitation device and controls for temperature, relative humidity, and sterilization.

Scaling-up is still a bottleneck for the widespread commercial application of SSF.

However, the development of rational and computer-controlled processes during

last decades brought about advances in SSF, namely: the modelling of microbial

growth on solid substrates, and energy and mass transfer in different types of

bioreactors. New methods are now available for measuring SSF parameters, such

as water activity and biomass production, as well as statistical tools for process

optimization. These breakthroughs in SSF will certainly promote the commercial

production and application of BCA.

WEST INDIAN JOURNAL

Paecilomyces lilacinus fungemia in a Jamaican neonate:=

Invasive opportunistic fungal infections have increased in frequency over the last two

decades, while Candida sp remains the most common cause of fungemia (1, 2). This

report describes the fourth documented case of Paecilomyces lilacinus fungemia, the

first such case to be reported at the University Hospital of the West Indies (UHWI).

A 35-week gestation female infant, weighing 3.5 kg was born to a 31-year old mother

with a normal antenatal history at a hospital in rural Jamaica. At birth, the infant was

jaundiced and was diagnosed as having Downs Syndrome. The infant subsequently

developed a low grade pyrexia and was transferred to the UHWI on day thirteen.

Significant findings included jaundice, pyrexia (100oF), abdominal distention and

cardiac murmurs consistent with a ventricular septal defect and patent ductus

arteriosus. A diagnosis of sepsis with necrotizing enterocolitis was made. Results of a

full septic screen were negative. A nasogastric tube was inserted for oral feeds and

intravenous ampicillin, cloxacillin, gentamicin and metronidazole were administered

through a peripheral intravenous catheter. These antibiotics were administered for

seven days with resolution of the fever and abdominal distention, and incomplete

resolution of jaundice. Oral feeds were subsequently commenced. The infant remained

mildly icteric and again developed abdominal distention and fever on day 14, when a

second septic screen was done. An ultrasound investigating the infants abdominal

distention revealed multiple abscesses, aspirates from which were culture positive for

Citrobacter diversus and Escherichia coli. A new antibiotic regimen was commenced

with intravenous rocephin, metronidazole and ampicillin. The infant however remained

febrile.

Results of the two sets of blood cultures taken from the peripheral vein on day 14 at the

UHWI were positive for a fungus initially identified as Penicillium spp. This isolate was

subsequently referred to Mayo Clinic, Minnesota, USA, for further identification.

Antimicrobial treatment was changed to include intravenous amphotericin B, amikacin

and ceftazidime. Amphotericin B was given for 18 days. The isolate was later confirmed

by Mayo Clinic to be Paecilomyces lilacinus The patient responded favourably to

treatment with amphotericin B and was discharged on maintenance fluconazole.

 

 

Paecilomyces lilacinus is a ubiquitous fungal saprophyte and an uncommon human

pathogen which is known to infect a variety of organs with varying degrees of morbidity

and mortality (1, 3, 4). Identification of the fungus is difficult as it is closely related to

the genus Penicillium (1). Prolonged use of antibiotics and invasive procedures,

including peripheral intravenous catheters predispose to acquisition of opportunistic

pathogens, such as Paecilomyces. Although Paecilomyces lilacinus has been reported to

be resistant to amphotericin B and fluconazole, the infant herein presented responded

favourably to this management (5).

Human pathogenicity:-

P. lilacinus is an infrequent cause of human disease. Most reported cases involve patients with compromised immune systems, indwelling foreign devices, or intraocular lens implants. Research of the last decade suggests it may be an emerging pathogen of both immunocompromised as well as immunocompetent adults.

Biocontrol agent:-

Plant-parasitic nematodes cause significant economic losses to a wide variety of crops. Chemical control is a widely used option for plant-parasitic nematode management. However, chemical nematicides are now being reappraised in respect of environmental hazard, high costs, limited availability in many developing countries or their diminished effectiveness following repeated applications.

Control of plant parasitic nematodes :-

P. lilacinus was first observed in association with nematode eggs in 1966 and the fungus was subsequently found parasitising the eggs of Meloidogyne incognita in Peru. It has now been isolated from many cyst and root-knot nematodes and from soil in many locations. Several successful field trials using P. lilacinus against pest nematodes were conducted in Peru. The Peruvian isolate was then sent to nematologists in 46 countries for testing, as part of the International Meloidogyne project, resulting in many more field trials on a range of crops in many soil types and climates Field trials, glasshouse trials and in vitro testing of P. lilacinus continues and more isolates have been collected from soil, nematodes and occasionally from insects. Isolates vary in their pathogenicity to plant-parasitic nematodes. Some isolates are aggressive parasites while other, though morphologically indistinguishable, are less or non-pathogenic. Sometimes isolates which looked promising in vitro or in glasshouse trials have failed to provide control in the field.

Enzymes :-

Many enzymes produced by P. lilacinus have been studied. A basic serine protease with biological activity against Meloidogyne hapla eggs has been identified. One strain of P. lilacinus has been shown to produce proteases and a chitinase, enzymes that could weaken a nematode egg shell so as to enable a narrow infection peg to push through.

Egg Infection :-

Before infecting a nematode egg, P. lilacinus flattens against the egg surface and becomes closely appressed to it. P. lilacinus produces simple appressoria anywhere on the nematode egg shell either after a few hyphae grow along the egg surface, or after a network of hyphae form on the egg. The presence of appressoria appears to indicate that the egg is, or is about to be, infected. In either case, the appressorium appears the same, as a simple swelling at the end of a hypha, closely appressed to the eggshell. Adhesion between the appressorium and nematode egg surface must be strong enough to withstand the opposing force produced by the extending tip of a penetration hypha When the hypha has penetrated the egg, it rapidly destroys the juvenile within, before growing out of the now empty egg shell to produce conidiophores and to grow towards adjacent eggs.

Crops :-Eggplant,Potato,Chilli,Tomatoes,Cucumbers, flowers, Orchards, Vineyards Ornamentals in greenhouses, lawns, nurseries and landscape.

Target Pests :-Plant parasitic nematodes in soil, Examples include Meloidogyne spp.(Root knot nematodes); Radopholus similis (Burrowing nematode); Heterodera spp. and Globodera spp. (Cyst nematodes); Pratylenchus spp. (Root lesion nematodes); Rotylenchulus reniformis (Reniform Nematode); Nacobbus spp.(False Root knot Nematodes).

Method of application :-

Paecilomyces lilacinus is a naturally occurring fungus found in many kinds of soils throughout the world. As a pesticide active ingredient, Paecilomyces lilacinus is applied to soil to control nematodes that attack plant roots. In laboratory studies, it grows optimally at 21-32 degrees C,and does not grow or survive above 36 degrees C. It acts against plant root nematodes by infecting eggs, juveniles, and adult females.

Suspend Paecilomyces lilacinus in sufficient water (500g/100L) to achieve uniform application. Apply at the rate of 100-200 g per cubic metre (loose) of greenhouse potting mix, soil or planting beds.

Paecilomyces lilacinus can be applied through low pressure watering nozzles such as fan nozzles or other watering systems (drip system) after filtering with filters. Agitate to maintain suspension. For best effect, treat potting mix several days before use for seeding or transplants.

Soil application :  For one acre mix about 3 kgs of Gmax Bioguard with 100 kgs of compost, keep the mixture under shade with sufficient moisture content (30%) for one week time and broadcast in the field.

Pit application: For plantation crops like Banana, sprinkle 25 grams of Gmax Bioguard in the pit before planting. After planting, about 25 grams of Gmax Bioguard can be mixed with compost and sprinkled around the tree trunk in the soil.

Frequency of application :-Two to three applications in vegetables ornamentals and 4-5 applications in lawns and landscape crops are recommended. In the case of high infestation multiple applications are

recommended. Applications during early stages of plant growth protect the plant during critical stages of development.

Dosage:-Soil application: 5 kg /ha along with any organic fertilizer (without pathogenic contaminants).Seed treatment: @ 4-5 gm per kg of seeds as per standard wet treatment.Seedling treatment: @ 100 g/l prior to planting.

(Sohwing normal xylem (NX) strands with hyphae(H) of Paecilomyces lilacinus).

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(Baarn: Centralbureau voor Schimmelcultures) 6: 119.

2. Anderson, Traute-Heidi; Domsch, K. H.; Gams, W. (1995). Compendium of Soil Fungi. Lubrecht & Cramer Ltd.

3. Marti GA, Lastra CC, Pelizza SA, García JJ (November 2006). "Isolation of Paecilomyces lilacinus (Thom) Samson (Ascomycota: Hypocreales) from the Chagas disease vector, Triatoma infestans Klug (Hemiptera: Reduviidae) in an endemic area in Argentina". Mycopathologia 162 (5): 369–72.

4. Fiedler, Z. and Sosnowska, D. (2007) Nematophagous fungus Paecilomyces lilacinus (Thom) Samson is also a biological agent for control of greenhouse insects and mite pests. BioControl 52, 547–558

5. Gupta, S.C., Leathers, T.D. and Wicklow, D.T. (1993) Hydrolytic enzymes secreted by Paecilomyces lilacinuscultured on sclerotia of Aspergillus flavus. Appl Microbiol Biotechnol 39, 99–103.

6. Saberhagen C, Klotz SA, Bartholomew W, Drews D, Dixon A (December 1997). "Infection due to Paecilomyces lilacinus: a challenging clinical identification". Clin. Infect. Dis. 25 (6): 1411–3.

7. Westenfeld F, Alston WK, Winn WC (June 1996). J. Clin. Microbiol. 34 (6): 1559–62.8. O'Day DM (January 1977). "Fungal endophthalmitis caused by Paecilomyces lilacinus after

intraocular lens implantation".9. Jatala, P; Kaltenbach R and Bocangel M. "Biological control of Meloidogyne incognita acrita and

Globodera pallida on potatoes". Journal of Nematology 11: 303.10. Stirling, GR (1991). Biological Control of Plant Parasitic Nematodes. UK: CABI Publishing.

pp. 282.11. Stirling, GR; West LM. "Fungal parasites of root-knot nematode eggs from tropical and sub-

tropical regions of Australia". Australasian Plant Pathology 20: 149–154.

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