Information

8.4: Fungal Pathogenicity - Biology


Learning Objectives

  • Name at least three fungal virulence factors that promote fungal colonization.
  • Name at least two fungal virulence factors that damage the host.

As with the bacteria, fungal virulence factors can be divided into two categories: virulence factors that promote fungal colonization of the host; and virulence factors that damage the host.

Virulence Factors that Promote Fungal Colonization

Virulence factors that promote fungal colonization of the host include the ability to:

1. adhere to host cells and resist physical removal;
2. invade host cells;
3. compete for nutrients;
4. resist innate immune defenses such as phagocytosis and complement; and
5. evade adaptive immune defenses.

Examples of virulence factors that promote fungal colonization include:

1. A compromised immune system is the primary predisposing factor for serious fungal infections. A person highly immunosuppressed, such as a person taking immunosuppressive drugs to suppress transplant rejection, or a person with advancing HIV infection, or a person with other immunosuppressive disorders, becomes very susceptible to infections by fungi generally considered not very harmful to a healthy person with normal defenses.

2. As with bacteria, the ability to adhere to host cells with cell wall adhesins seems to play a role in fungal virulence.

3. Some fungi produce capsules allowing them to resist phagocytic engulfment, such as the yeast Cryptococcus neoformans and the yeast form of Histoplasma capsulatum (Figure (PageIndex{1})).

4. Candida albicans stimulates the production of a cytokine called GM-CSF and this cytokine can suppress the production of complement by monocytes and macrophages. This may decrease the production of the opsonin C3b as well as the complement proteins that enhance chemotaxis of phagocytes.

5. C. albicans also appears to be able to acquire iron from red blood cells.

6. albicans produces acid proteases and phospholipases that aid in the penetration and damage of host cell membranes.

7. Some fungi are more resistant to phagocytic destruction, e.g., Candida albicans, Histoplasma capsulatum, and Coccidioides immitis.

8. There is evidence that when the yeast form of Candida enters the blood it activates genes allowing it to switch from its budding form to its hyphal form. In addition, when engulfed by macrophages, it starts producing the tubular germ tubes which penetrate the membrane of the macrophage thus causing its death.

A movie of Candida killing a macrophage from within from the Theriot Lab Website at Stanford University Medical School: Candida albicans killing macrophages from inside out.

9. Factors such as body temperature, osmotic stress, oxidative stress, and certain human hormones activate a dimorphism-regulating histidine kinase enzyme in dimorphic molds, such as Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis, causing them to switch from their avirulent mold form to their virulent yeast form. It also triggers the yeast Candida albicans to switch from its yeast form to its more virulent hyphal form.

Virulence Factors that Damage the Host

Like bacteria, fungal PAMPs binding to PRRs can trigger excessive cytokine production leading to a harmful inflammatory response that damages tissues and organs. As fungi grow in the body, they can secrete enzymes to digest cells. These include proteases, phospholipases, and elastases. In response to both the fungus and to cell injury, cytokines are released. As seen earlier under Bacterial Pathogenesis, this leads to an inflammatory response and extracellular killing by phagocytes that leads to further destruction of host tissues.

Many molds secrete mycotoxins , especially when growing on grains, nuts and beans. These toxins may cause a variety of effects in humans and animals if ingested including loss of muscle coordination, weight loss, and tremors. Some mycotoxins are mutagenic and carcinogenic. Aflatoxins, produced by certain Aspergillus species, are especially carcinogenic. A mold called Stachybotrys chartarum is a mycotoxin producer that has been implicated as a potential serious problem in homes and buildings as one of the causes of "sick building syndrome." Mycotoxin symptoms in humans include dermatitis, inflammation of mucous membranes, , cough, fever, headache, and fatigue.

Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.

  • Candida albicans
  • Cryptococcus neoformans
  • Pneumocystis carinii
  • Dermatophytic infections (tinea)
  • Coccidioides immitis
  • Histoplasma capsulatum
  • Blastomyces dermatitidis
  • Aspergillosis
  • Rhizopus
  • Mold allergy

Summary

Many of the same factors that enable bacteria to colonize the body also enable fungi to colonize. Many of the same factors that enable bacteria to harm the body also enable fungi to cause harm.


Pathogenic Fungus

Pathogenic fungi make people and other organisms sick and can kill them. For humans, about 300 pathogenic species of fungi are known. Some of them are Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys. One example of Cryptococcus is Cryptococcus neoformans which causes severe meningitis in people who are infected with HIV or have AIDS.

The skin, gastrointestinal tract, respiratory tract and genital-urinary tract are areas of the body that often become infected with pathogenic fungi. People with higher levels of monocytes/macrophages, invariant natural killer (iNK) T-cells and dendritic cells have a better chance of controlling a fungus infection and preventing it from spreading throughout the body.


The image above shows Aspergillus terreus. This pathogenic fungus causes infections of the ears, skin, nails and lungs of people with compromised immune systems.


Background

In the past 25 years, the opportunistic human pathogen Candida albicans has become a serious medical problem. This fungus is now fourth on the list of hospital-acquired infections, ahead of Gram-negative bacteria, and despite the recent introduction of a new class of antifungals, drug resistance continues to be a problem [1]. In the past 15 years, molecular techniques have been applied to understand the pathogenesis of this organism as well as to search for novel drug targets. However, C. albicans presents several difficulties for molecular biologists: it is diploid only a part of a sexual cycle has been demonstrated it has a very plastic genome and it is highly heterozygous. Each of these properties is best investigated through a genomic approach. Hence, knowledge of the genome sequence has been an important goal for the past 10 years. More recently, genome structure and dynamics have become increasingly important in this organism as widespread aneuploidy [2, 3], the role of repeated DNA in chromosome loss [4], and chromosome rearrangement leading to drug resistance [5] have been reported.

The Candida Genome Sequencing Project started in 1996 and, in 2004, it produced a diploid assembly constructed from 10.9× coverage (Assembly 19), which provided single contigs where heterozygosity was not obvious and allelic contigs where there was significant heterozygosity [6]. There were several important steps along the way to this release these are detailed in a review by Nantel [7]. The first was the construction of a physical map of one chromosome, chromosome 7 [8]. Next were the two early releases of the emerging sequence data, called Assembly 4 and Assembly 6. These lower density assemblies facilitated a great deal of gene analysis, including the construction of several microarrays [9, 10], an analysis of haploinsufficient genes for filamentation [11], and the elucidation of several gene families, including a number important in pathogenesis. Examples include the secreted aspartyl proteinases (SAPs) [12], the agglutinin-like substances (ALSs) [13], and the phospholipases (PLB and PLC) [14, 15]. Two quite comprehensive disruption libraries are currently available. One library was constructed systematically by targeted disruption of one allele followed by insertion of a regulated promoter at the other allele [16]. The other disruption library was constructed randomly by transposon mutagenesis, using as an insert into one allele the UAU cassette, which facilitates disruption of the second allele via two spontaneously occurring steps of mitotic recombination [17].

These tools have greatly advanced the pace of molecular analysis of the pathogenesis and life style of C. albicans, but Assembly 19 was not a finished sequence, since it contained a total of 412 contigs, of which 266 were the haploid set. In order to provide a finished sequence, we used hybridization of chromosomes partially purified by pulse-field elecrophoresis as well as a sequence tagged site (STS) map based on a fosmid library to identify the chromosomal location of various contigs. We then utilized bioinformatics to analyze both the emerging sequence of C. albicans strain WO-1, the sister species Candida dubliniensis and the primary traces used to generate Assembly 4, and coupled this with the STS map and a whole-chromosome optical map to construct Assembly 21. This assembly has eight linear DNA sequences including nine copies of the intermediate repeat called the major repeat sequence (MRS), of which three have been completely sequenced. The MRS is made up of three subrepeats, called RB2, RPS, and HOK [18]. In addition to the intact MRS sequences, there are 14 RB2 sequences and 2 HOK sequences. The ribosomal DNA constitutes another repeat, which is not included in the assembly. In addition to its usefulness for gene mapping, Assembly 21 reveals some interesting biological features, including a putative transcription factor gene family with members proximal to 14 of the 16 telomeres, a telomere-like sequence in the middle of chromosome 1, information on the relationships of chromosome location to similarity of gene families, and a revised open reading frame (ORF) list.


Introduction

I nvasive fungal infections (IFIs) are caused by opportunistic fungi such as the filamentous Aspergillus fumigatus or the yeasts Candida albicans and Cryptococcus neoformans (Enoch et al., 2006). Though not typically a concern in healthy individuals, IFIs are able to afflict ill or immunocompromised patients severely, including individuals with leukemia, transplant recipients, and those with HIV/AIDS (Comely et al., 2015 de Oliveira et al., 2014 Klingspor et al., 2015 Neofytos et al., 2013).

The incidence of IFIs is increasing, and a large proportion of these IFIs are nosocomial (Beck-Sagué and Jarvis, 1993 Lehrnbecher et al., 2010). This is believed to be due to an increase in the population of immunocompromised individuals ( Lehrnbecher et al., 2010 Warnock, 2007). IFIs tend to have high mortality rates (Comely et al., 2015 Lehrnbecher et al., 2010), and as a result the improvement of current prophylactic and curative treatments is of increasing interest. It is essential that we understand the fundamental and dynamic biological interactions between host and fungal cells in order to advance the care and treatment of patients with IFIs.

Pathogenesis requires an interaction between a pathogen and its host. There are numerous examples of host–fungal interactions in the context of organisms causing IFIs. Aspergillus fumigatus has been shown to adhere to extracellular matrix of the lung as well as the surface of human lung epithelial cells (Gil et al., 1996, Sheppard, 2011). Additionally, the internalization of A. fumigatus spores by epithelial cells in vitro has been observed numerous times (Gomez et al., 2010 Oosthuizen et al., 2011 Wasylnka and Moore, 2003).

Candida albicans has been observed to invade host cells by inducing endocytosis (Dalle et al., 2010) or through active invasion, a process by which hyphae breach epithelial cell membranes (Dalle et al., 2010, Wächtler et al., 2011). It has been demonstrated that C. neoformans infects its host through an actin-dependent internalization mechanism (Guerra et al., 2014). These initial interactions often lead to other interactions between the host and fungus on numerous levels. Host–fungal interaction networks are extremely complex, as there are many inherent differences between mammalian cells and fungal cells. A comprehensive analysis of these networks would entail the use of “-omics”-wide techniques in order to capture both the drastic and the subtle dynamic biological perturbations within both host and pathogen.

The study of various biological “-omics” is generally segregated into several major fields of high-throughput biology, notably genomics, transcriptomics, proteomics, and metabolomics. An ideal -omic analysis of an organism involves collection of complete and unbiased datasets representative of the entire set of biomolecules of interest. Techniques that do not select specific, or candidate, targets are of particular value as they permit identification of novel biological networks without prior knowledge. The use of high-throughput techniques such as these has recently become far more commonplace as they can provide a more complete picture of the complexities of an organism's or cell's responses to experimental or environmental conditions. More prevalent quantitative techniques such as western blots and reverse transcription quantitative PCR are only able to analyze specific targets and are thus unable to detect unexpected changes.

Historically, high-throughput biology has been associated with a prohibitive monetary cost, rendering many of these techniques inaccessible to most researchers. Despite this, high-throughput biology has undeniable potential for the systematic analysis of a complex biological system, such as a host–pathogen interaction (Fig. 1). Researcher uptake has been aided by an increase in affordability of several high-throughput biology techniques in recent years. A notable example is the price of commercial genome sequencing, with the full sequencing of a human genome now as low as US $1000 (Veritas Genomics, 2015).

FIG. 1. Systems biology of dual-organism interactions. A defined experimental host–pathogen system is analyzed using high-throughput methods. Collected data are subjected to computational statistical analysis, and the results are analyzed using a number of bioinformatics technologies. Data analysis yields a model for the biological interaction. Replication results in a robust and validated model for the biological system. This validated model is then used to determine aspects of the system requiring further study and refinement.

There are previous reviews on topics related to the host–fungal interaction (Durmuş et al., 2015 Horn et al., 2012 Santamaría et al., 2011), and our review builds upon these by outlining and integrating both experimental and computational methods in high-throughput biology. We place particular emphasis on recent innovations in technology that promise to yield valuable insights into the relatively limited field of host–fungal interactions.


Identification and Pathogenicity of Fungal Pathogens Associated with Stem End Rots of Avocado Fruits in Kenya

Losses associated with stem end rot (SER) of avocado fruits have been reported in all avocado growing regions of the world. In Kenya, mature avocado fruits present SER symptoms during storage and marketing, but the disease causal agent(s) has not been established. This study aimed to identify the fungal pathogen(s) associated with avocado SER in Kenya and evaluate its pathogenicity. Fungal isolates were collected from symptomatic avocado fruits from randomly selected orchards and major markets within Murang'a County, a major avocado growing region in Kenya, between September 2017 and March 2018. A total of 207 and 125 fungal isolates, recovered from orchards and major markets, respectively, were identified morphologically and further confirmed by molecular techniques. The identified isolates were Lasiodiplodia theobromae (39.8%), Neofusicoccum parvum (24.4%), Nectria pseudotrichia (18.4%), Fusarium solani (7.2%), F. oxysporum (5.1%), F. equiseti (3.9%), and Geotricum candidum (1.2%). Geotricum candidum was exclusively recovered from fruits from the market. In the pathogenicity test, L. theobromae, N. parvum, and N. pseudotrichia caused the most severe SER symptoms. Consequently, they were considered to be the major pathogens of SER of avocado fruits in Kenya. To our knowledge, this is the first report of SER pathogen of avocado fruits in Kenya. Given the significant contribution of avocado fruits to household income and foreign exchange in Kenya, this information is significant to further develop management strategies of postharvest loss of avocado fruits in Kenya.

1. Introduction

In Kenya, avocado (Persea americana Mill.) is one of the most important perennial tropical fruit crops and a major foreign exchange earner. In 2017, it accounted for about 74% by value of the total fruits exported from the country [1]. Currently, “Hass” avocado contributes approximately 80% of the avocado fruits produced and exported from Kenya [2]. Other cultivars produced include “Fuerte,” “Puebla,” “Duke,” and “G6” [3]. Avocado production in Kenya is dominated by smallholder farmers (85%) within several agroecological zones, who mainly produce for the export market, and the remainder is sold in the local markets. Seventy percent (70%) of the avocado fruits are produced in the central and eastern regions of the country. The fruits are exported mainly to the European Union [2, 4]. Since the year 2000, the acreage under avocado production has increased significantly, leading to increased export of the avocado fruit from Kenya [4]. The increased production is fueled by high demand for avocado fruits in the global market due to consumer awareness of the dietary value of the fruit [5]. Despite the increased production and export of avocado fruits from Kenya, high incidences of postharvest fungal diseases, including anthracnose and SER, limit marketing of the fruits and contribute to increased losses by the producers [6, 7].

The symptoms of stem end rot (SER) develop on the avocado fruit as it ripens. It is characterized by shriveling, followed by brown to black rot that starts at the stem end of the fruit. As the rot progresses, internal vascular bundles may have black to brown colorations and eventually the whole fruit is consumed by the rot [8, 9]. Fruits hardly display SER symptoms before harvest. Furthermore, SER often occur at the packing house during transit or after marketing.

Various fungal species have been reported to cause SER on avocado fruits. In Chile, the fungal pathogens reported to cause SER included members of Botryosphaeriaceae family, namely Diplodia mutila, D. pseudoseriata, D. seriata, Dothiorella iberica, Lasiodiplodia theobromae, Neofusicoccum australe, N. nonquaesitum, and N. parvum [10]. In Italy, N. parvum, Colletotrichum gloeosporioides, or C. fructicola and Diaporthe foeniculacea or D. sterilis were the most isolated SER pathogens [9]. In California, Neofusicoccum luteum and Phomopsis perseae were reported [8] while in South Africa, Thyronectria pseudotrichia, Dothiorella aromatica, Pestalotiopsis versicolor, Lasiodiplodia theobromae, Rhizopus stolonifer, Fusarium sambucinum, and Fusarium solani were reported [11].

In Kenya, however, the actual pathogen causing SER has not been identified, but on the other hand, anthracnose pathogens have been described [12]. Therefore, this study aimed at identifying the fungal pathogen associated with SER of avocado fruits in the central highlands of Kenya and testing their pathogenicity.

2. Materials and Methods

2.1. Study Area and Sample Collection

The study was conducted in Murang'a County, which is the leading county in production and export of avocado fruits in Kenya [1]. Geographically, the county lies between latitudes 0°34′ south and 1°07′ south and longitudes 36° east and 37°27′ east, with an elevation of 914 m a.s.l in the east and 3,353 m a.s.l in the west. Avocado fruits are cultivated in the agroecological zones two, three, and four that have 18.0°C to 27.2°C average temperature ranges and 1600 mm–900 mm average annual rainfall [13].

Between September 2017 and March 2018, systematic sampling was used to select 162 orchards included in the study. The orchards had more than five “Hass” avocado fruit trees. Six mature avocado fruits were harvested at random from each five randomly selected avocado fruit trees in every sampled orchard. In addition, 10 “Hass” fruits, at different stages of ripening, were bought from different traders in three major markets (Kandara, Kirwara, and Maragwa) within the county at weekly intervals for two months. A total of 453 fruits from 4,860 fruits harvested from the orchards and 240 fruits from the market were sampled, packed in cartons, and transported to Kenya Agricultural and Livestock Research Organization (KALRO), Kandara, where they were incubated at room temperature (22°C–25°C) for 7–14 days to allow development of SER.

2.2. Fungal Isolation

The 207 fruits from the orchards and 125 fruits from the market that displayed SER symptoms were washed with clean tap water, surface-sterilized with 2% sodium hypochlorite for one minute, rinsed in distilled water, and air-dried. Small pieces of flesh from the margins of symptomatic flesh were placed aseptically in 9 cm diameter Petri dishes containing potato dextrose agar (PDA) amended with streptomycin sulfate and incubated at room temperature (22°C–25°C) for five days. Pure cultures were obtained by transferring the mycelia tips on 1.5% (wt/vol) water agar (WA) and allowed to grow overnight. Hyphal tips of the mycelia growth in the WA were later transferred onto PDA amended with streptomycin sulfate. Slant universal bottle was used to preserve the pure cultures of the pathogen and stored in the fridge at 4°C for later use.

2.2.1. Preparation of Conidial Suspension

Fourteen-day-old pure cultures in PDA were flooded with sterile distilled water. A sterile wire loop was used to scrape off the conidia and bring them to suspension. The suspension was filtered through a double-layer muslin cloth and the collected filtrate diluted serially to 1 × 10 5 . A haemocytometer was used to adjust the spore concentration.

2.3. Morphological Characterization of the Isolate

To induce conidia production, small pieces of mycelia from the isolates were transferred into 9 cm diameter Petri dishes with PDA amended with autoclaved avocado wood chips and incubated at 25 ± 1°C for four weeks. The isolates were morphologically identified based on cultural and microscopic characteristics as described by Valencia et al. [10], Phillips et al. [14], and Watanabe [15]. Lactophenol blue was used in microscopic identification. The length and width of conidia (N = 50) from each isolate were measured using light microscope Zeiss-Primo Star, coupled to AxioCam ERc 5s camera.

2.4. Molecular Characteristics
2.4.1. DNA Extraction

An improved fungal extraction protocol described by Innis et al. [16] was used to extract DNA from three representative isolates of each species. Pure fungal cultures derived from the single spores incubated in PDA were used. Forty milligram (mg) of mycelium was placed in a microcentrifuge tube containing 300 μl of extraction buffer (Tris-HCl, 200 mM Ph 8.5 EDTA, 25 mM 1 M NaCl 250 mM SDS, 0.5%) with glass beads. The tubes were placed in a fastprep®-24 genogrinder for one minute at 2000 rpm. Two hundred microlitre (μl) of 3 mM sodium acetate pH5.2 was added and refrigerated at −20°C for 10 minutes. After incubation, the samples were centrifuged for 10 minutes at 13000 rpm. After that, the supernatants were transferred into fresh 1.5 ml microcentrifuge tubes. Equal amounts of isopropanol were added to the supernatants and allowed to stand for five minutes at room temperature. After five minutes, the samples were centrifuged for 10 minutes at 13000 rpm and the supernatant was discarded. Five hundred μl of 70% ethanol was then added to the pellets and centrifuged at 13000 rpm for 10 minutes to wash the pellet. The nucleic acid pellets obtained were air-dried and then resuspended in 50 μl of low salt TE buffer (Tris-HCl, 1 mM, pH 8 EDTA, 0.1 mM) and stored at −20°C for later use. The quality of DNA was determined by agarose gel electrophoresis and quantified using a NanoDrop ND-1000Spectrophotometer. DNA was standardized or normalized to 20 ng/μl for polymerase chain reactions (PCR).

2.4.2. DNA Amplification and Sequencing

The extracted DNA was used as templates in PCR. Two sets of primers, ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCC TCC GCT TAT TGA TAT GC), ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS1, were used in the amplification of the internal transcribed region rDNA of the fungal isolates [16]. PCR reaction volumes of 25 μl containing 2.5 μl of 0.2 μΜ of each primer, 5 × My Taq reaction buffer, 0.25 μl Taq polymerase (Bioline, Meridian Life Science, Memphis, USA), 40 ng/μl of each DNA template, and 12.75 μl of molecular water were used. For amplification, the GeneAmp 9700 DNA Thermal Cycler (Perkin-Elmer) was used. The process involved an initial denaturing step at 94°C for 30 s, followed by 35 cycles, denaturing at 94°C for 30 s, annealing at 55°C for 30 s followed by extension for 1 minute at 68°C, and a final extension step of 5 min at 68°C. To confirm amplification, the PCR products were run on 1.5% agarose gel and visualized under UV light using ENDURO™ GDS. The PCR products were cleaned using the Qiagen PCR cleaning kit according to manufacturer instructions and submitted for Sanger sequencing with forward and reverse primers at Inqaba Africa Genomic platform, South Africa.

2.4.3. Bioinformatics Analysis

Sequence data was analyzed by assigning reads to samples, indexes, primers, and adapters. The primers were marked using Picard (https://broadinstitute.github.io/picard/index.html). Bam2fastq (https://gsl.hudsonalpha.org/information/software/bam2fastq) was used to convert the resultants’ bam files to fastq. The overall sequencing quality of the reads was evaluated visually using the Fast QC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The quality parameters used in filtering the reads included a minimum length of 250 bp and a minimum QC value of 30. Trimming was done corresponding to the adapters and low-quality sequences from all the reads. Subsequent analysis and processing of the reads was done in the CLC Genomic Workbench 11.0, where the overlapping reads were merged. The de novo assembly of the unassembled reads and the raw reads’ alignment was performed using CLC Genomics Workbench 11.0 with default parameters (minimum contig = 100 bp, 23 K-mer, similarity fraction = 80%, and length fraction = 50%). BLAST analysis of the ITS sequences was done to support the morphological identification of the samples at the NCBI database. The ITS sequences were deposited in GeneBank using BankIT.

2.5. Pathogenicity Test

To establish Koch’s postulates, Geotrichum candidum, Fusarium equiseti, Fusarium oxysporum, Fusarium solani, Lasiodiplodia theobromae, Neofusicoccum parvum, and Nectria pseudotrichia were subjected to pathogenicity test as described by Freeman et al. [17] and Twizeyimana et al. [8]. Healthy “Hass” fruits were harvested from the farms known to have a low incidence of SER within Murang'a County. The fruits were washed with clean tap water to remove any soil debris. The fruits were surface-sterilized by dipping in 75% ethanol for about three minutes, rinsed with distilled water, and then air-dried. Each of the isolates was subjected to two methods of inoculation.

A sterile cork borer (5 mm diameter) was used to wound the stem end of each fruit and mycelial discs of equivalent diameter obtained from the edge of actively growing pure cultures were placed on the wound. Six inoculated fruits for each pathogen and six control fruits inoculated with plain PDA were arranged on individual trays and covered with cling film to conserve moisture and avoid contamination. The fruits were incubated at room temperature of 24°C ± 1.

After snapping the pedicel of air-dried fruits, conidial suspension (5 × 10 −5 conidial/ml) was placed on stem end opening and covered with cling film. Six inoculated fruits for each pathogen and six control fruits inoculated with distilled water were arranged in individual trays and covered with cling film. The inoculated fruits were incubated at 24 ± 1°C. Evaluation was done after 12 days by cutting the fruits longitudinally and rating SER symptoms on a 0–4 rating scale as follows: 0 = no visible rot 1 = 1–25% rot 2 = 25–50% rot 3 = 50–75% rot 4 = ≥75% rot (Figure 1). At the end of the pathogenicity test, reisolation from the symptomatic fruits was made, and reisolated fungal colonies compared morphologically to the original isolates [8, 9]. SER severity on avocado fruits was calculated using the following formula [18]:


8.4: Fungal Pathogenicity - Biology

From crop and food spoilage to severe infections in animal species, fungal parasites and pathogens are wide spread and difficult to treat.

Learning Objectives

Give examples of fungi that are plant and animal parasites and pathogens

Key Takeaways

Key Points

  • In plants, fungi can destroy plant tissue directly or through the production of potent toxins, which usually ends in host death and can even lead to ergotism in animals like humans.
  • During mycosis, fungi, like dermatophytes, successfully attack hosts directly by colonizing and destroying their tissues.
  • Examples of fungal parasites and pathogens in animals that cause mycoses include Batrachochytrium dendrobatidis, Geomyces destructans, and Histoplasma capsulatum.
  • Systemic mycoses, such as valley fever, Histoplasmosis, or pulmonary disease, are fungal diseases that spread to internal organs and commonly enter the body through the respiratory system.
  • Opportunistic mycoses, fungal infections that are common in all environments, mainly take advantage of individuals who have a compromised immune system, such as AIDS patients.
  • Fungi can also cause mycetismus, a disease caused by the ingestion of toxic mushrooms that leads to poisoning.

Key Terms

  • mycosis: a fungal disease caused by infection and direct damage
  • dermatophyte: a parasitic fungus that secretes extracellular enzymes that break down keratin, causing infections the skin, such as jock itch and athlete’s foot
  • aflatoxin: toxic, carcinogenic compounds released by fungi of the genus Aspergillus contaminate nut and grain harvests
  • ergot: any fungus in the genus Claviceps which are parasitic on grasses

Fungal Parasites and Pathogens

The production of sufficient good-quality crops is essential to human existence. Plant diseases have ruined crops, bringing widespread famine. Many plant pathogens are fungi that cause tissue decay and eventual death of the host. In addition to destroying plant tissue directly, some plant pathogens spoil crops by producing potent toxins. Fungi are also responsible for food spoilage and the rotting of stored crops. For example, the fungus Claviceps purpurea causes ergot, a disease of cereal crops (especially of rye). Although the fungus reduces the yield of cereals, the effects of the ergot’s alkaloid toxins on humans and animals are of much greater significance. In animals, the disease is referred to as ergotism. The most common signs and symptoms are convulsions, hallucinations, gangrene, and loss of milk in cattle. The active ingredient of ergot is lysergic acid, which is a precursor of the drug LSD. Smuts, rusts, and powdery or downy mildew are other examples of common fungal pathogens that affect crops.

Fungal pathogens: Some fungal pathogens include (a) green mold on grapefruit, (b) powdery mildew on a zinnia, (c) stem rust on a sheaf of barley, and (d) grey rot on grapes.

Aflatoxins are toxic, carcinogenic compounds released by fungi of the genus Aspergillus. Periodically, harvests of nuts and grains are tainted by aflatoxins, leading to massive recall of produce. This sometimes ruins producers and causes food shortages in developing countries.

Animal and Human Parasites and Pathogens

Fungi can affect animals, including humans, in several ways. A mycosis is a fungal disease that results from infection and direct damage. Fungi attack animals directly by colonizing and destroying tissues. Mycotoxicosis is the poisoning of humans (and other animals) by foods contaminated by fungal toxins (mycotoxins). Mycetismus describes the ingestion of preformed toxins in poisonous mushrooms. In addition, individuals who display hypersensitivity to molds and spores develop strong and dangerous allergic reactions. Fungal infections are generally very difficult to treat because, unlike bacteria, fungi are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the eukaryotic animal host.

Many fungal infections are superficial that is, they occur on the animal’s skin. Termed cutaneous (“skin”) mycoses, they can have devastating effects. For example, the decline of the world’s frog population in recent years may be caused by the fungus Batrachochytrium dendrobatidis, which infects the skin of frogs and presumably interferes with gaseous exchange. Similarly, more than a million bats in the United States have been killed by white-nose syndrome, which appears as a white ring around the mouth of the bat. It is caused by the cold-loving fungus Geomyces destructans, which disseminates its deadly spores in caves where bats hibernate. Mycologists are researching the transmission, mechanism, and control of G. destructans to stop its spread.

Fungi that cause the superficial mycoses of the epidermis, hair, and nails rarely spread to the underlying tissue. These fungi are often misnamed “dermatophytes”, from the Greek words dermis meaning skin and phyte meaning plant, although they are not plants. Dermatophytes are also called “ringworms” because of the red ring they cause on skin. They secrete extracellular enzymes that break down keratin (a protein found in hair, skin, and nails), causing conditions such as athlete’s foot and jock itch. These conditions are usually treated with over-the-counter topical creams and powders they are easily cleared. More persistent superficial mycoses may require prescription oral medications.

Mycosis infection: (a) Ringworm presents as a red ring on skin (b) Trichophyton violaceum, shown in this bright field light micrograph, causes superficial mycoses on the scalp (c) Histoplasma capsulatum is an ascomycete that infects airways and causes symptoms similar to influenza.

Systemic mycoses spread to internal organs, most commonly entering the body through the respiratory system. For example, coccidioidomycosis (valley fever) is commonly found in the southwestern United States where the fungus resides in the dust. Once inhaled, the spores develop in the lungs and cause symptoms similar to those of tuberculosis. Histoplasmosis is caused by the dimorphic fungus Histoplasma capsulatum. It also causes pulmonary infections. In rarer cases, it causes swelling of the membranes of the brain and spinal cord. Treatment of these and many other fungal diseases requires the use of antifungal medications that have serious side effects.

Opportunistic mycoses are fungal infections that are either common in all environments or are part of the normal biota. They mainly affect individuals who have a compromised immune system. Patients in the late stages of AIDS suffer from opportunistic mycoses that can be life threatening. The yeast Candida sp., a common member of the natural biota, can grow unchecked and infect the vagina or mouth (oral thrush) if the pH of the surrounding environment, the person’s immune defenses, or the normal population of bacteria are altered.

Mycetismus can occur when poisonous mushrooms are eaten. It causes a number of human fatalities during mushroom-picking season. Many edible fruiting bodies of fungi resemble highly-poisonous relatives. Amateur mushroom hunters are cautioned to carefully inspect their harvest and avoid eating mushrooms of doubtful origin.


Contents

Control of plant diseases is crucial to the reliable production of food, and it provides significant problems in agricultural use of land, water, fuel and other inputs. Plants in both natural and cultivated populations carry inherent disease resistance, but there are numerous examples of devastating plant disease impacts such as the Great Famine of Ireland and chestnut blight, as well as recurrent severe plant diseases like rice blast, soybean cyst nematode, and citrus canker.

However, disease control is reasonably successful for most crops. Disease control is achieved by use of plants that have been bred for good resistance to many diseases, and by plant cultivation approaches such as crop rotation, use of pathogen-free seed, appropriate planting date and plant density, control of field moisture, and pesticide use. Continuing advances in the science of plant pathology are needed to improve disease control, and to keep up with changes in disease pressure caused by the ongoing evolution and movement of plant pathogens and by changes in agricultural practices.

Plant diseases cause major economic losses for farmers worldwide. Across large regions and many crop species, it is estimated that diseases typically reduce plant yields by 10% every year in more developed settings, but yield loss to diseases often exceeds 20% in less developed settings. The Food and Agriculture Organization estimates that pests and diseases are responsible for about 25% of crop loss. To solve this, new methods are needed to detect diseases and pests early, such as novel sensors that detect plant odours and spectroscopy and biophotonics that are able to diagnose plant health and metabolism. [2]

In most pathosystems, virulence is dependent on hydrolases - and the wider class of cell wall degrading proteins - that degrade the cell wall. The vast majority of CWDPs are pathogen-produced and pectin-targeted (for example, pectinesterase, pectate lyase, and pectinases). For microbes the cell wall polysaccharides are themselves a food source, but mostly just a barrier to be overcome.

Many pathogens also grow opportunistically when the host breaks down its own cell walls, most often during fruit ripening. [3]

Fungi Edit

Most phytopathogenic fungi belong to the Ascomycetes and the Basidiomycetes. The fungi reproduce both sexually and asexually via the production of spores and other structures. Spores may be spread long distances by air or water, or they may be soilborne. Many soil inhabiting fungi are capable of living saprotrophically, carrying out the part of their life cycle in the soil. These are facultative saprotrophs. Fungal diseases may be controlled through the use of fungicides and other agriculture practices. However, new races of fungi often evolve that are resistant to various fungicides. Biotrophic fungal pathogens colonize living plant tissue and obtain nutrients from living host cells. Necrotrophic fungal pathogens infect and kill host tissue and extract nutrients from the dead host cells. Significant fungal plant pathogens include: [ citation needed ]

Ascomycetes Edit

  • Fusarium spp. (Fusarium wilt disease)
  • Thielaviopsis spp. (canker rot, black root rot, Thielaviopsis root rot)
  • Verticillium spp.
  • Magnaporthe grisea (rice blast)
  • Sclerotinia sclerotiorum (cottony rot)

Basidiomycetes Edit

  • Ustilago spp. (smuts) smut of barley
  • Rhizoctonia spp.
  • Phakospora pachyrhizi (soybean rust)
  • Puccinia spp. (severe rusts of cereals and grasses)
  • Armillaria spp. (honey fungus species, virulent pathogens of trees)

Fungus-like organisms Edit

Oomycetes Edit

The oomycetes are fungus-like organisms. [4] They include some of the most destructive plant pathogens including the genus Phytophthora, which includes the causal agents of potato late blight [4] and sudden oak death. [5] [6] Particular species of oomycetes are responsible for root rot.

Despite not being closely related to the fungi, the oomycetes have developed similar infection strategies. Oomycetes are capable of using effector proteins to turn off a plant's defenses in its infection process. [7] Plant pathologists commonly group them with fungal pathogens.

Significant oomycete plant pathogens include:

Phytomyxea Edit

Some slime molds in Phytomyxea cause important diseases, including club root in cabbage and its relatives and powdery scab in potatoes. These are caused by species of Plasmodiophora and Spongospora, respectively.

Bacteria Edit

Most bacteria that are associated with plants are actually saprotrophic and do no harm to the plant itself. However, a small number, around 100 known species, are able to cause disease. [8] Bacterial diseases are much more prevalent in subtropical and tropical regions of the world.

Most plant pathogenic bacteria are rod-shaped (bacilli). In order to be able to colonize the plant they have specific pathogenicity factors. Five main types of bacterial pathogenicity factors are known: uses of cell wall–degrading enzymes, toxins, effector proteins, phytohormones and exopolysaccharides.

Pathogens such as Erwinia species use cell wall–degrading enzymes to cause soft rot. Agrobacterium species change the level of auxins to cause tumours with phytohormones. Exopolysaccharides are produced by bacteria and block xylem vessels, often leading to the death of the plant.

Bacteria control the production of pathogenicity factors via quorum sensing.

Significant bacterial plant pathogens:

Phytoplasmas and spiroplasmas Edit

Phytoplasma and Spiroplasma are genera of bacteria that lack cell walls and are related to the mycoplasmas, which are human pathogens. Together they are referred to as the mollicutes. They also tend to have smaller genomes than most other bacteria. They are normally transmitted by sap-sucking insects, being transferred into the plant's phloem where it reproduces.

Viruses, viroids and virus-like organisms Edit

There are many types of plant virus, and some are even asymptomatic. Under normal circumstances, plant viruses cause only a loss of crop yield. Therefore, it is not economically viable to try to control them, the exception being when they infect perennial species, such as fruit trees.

Most plant viruses have small, single-stranded RNA genomes. However some plant viruses also have double stranded RNA or single or double stranded DNA genomes. These genomes may encode only three or four proteins: a replicase, a coat protein, a movement protein, in order to allow cell to cell movement through plasmodesmata, and sometimes a protein that allows transmission by a vector. Plant viruses can have several more proteins and employ many different molecular translation methods.

Plant viruses are generally transmitted from plant to plant by a vector, but mechanical and seed transmission also occur. Vector transmission is often by an insect (for example, aphids), but some fungi, nematodes, and protozoa have been shown to be viral vectors. In many cases, the insect and virus are specific for virus transmission such as the beet leafhopper that transmits the curly top virus causing disease in several crop plants. [11] One example is mosaic disease of tobacco where leaves are dwarfed and the chlorophyll of the leaves is destroyed. Another example is Bunchy top of banana, where the plant is dwarfed, and the upper leaves form a tight rosette.

Nematodes Edit

Nematodes are small, multicellular wormlike animals. Many live freely in the soil, but there are some species that parasitize plant roots. They are a problem in tropical and subtropical regions of the world, where they may infect crops. Potato cyst nematodes (Globodera pallida and G. rostochiensis) are widely distributed in Europe and North and South America and cause $300 million worth of damage in Europe every year. Root knot nematodes have quite a large host range, they parasitize plant root systems and thus directly affect the uptake of water and nutrients needed for normal plant growth and reproduction, [12] whereas cyst nematodes tend to be able to infect only a few species. Nematodes are able to cause radical changes in root cells in order to facilitate their lifestyle.

Protozoa and algae Edit

There are a few examples of plant diseases caused by protozoa (e.g., Phytomonas, a kinetoplastid). [13] They are transmitted as durable zoospores that may be able to survive in a resting state in the soil for many years. Further, they can transmit plant viruses. When the motile zoospores come into contact with a root hair they produce a plasmodium which invades the roots.

Some colourless parasitic algae (e.g., Cephaleuros) also cause plant diseases. [ citation needed ]

Parasitic plants Edit

Parasitic plants such as broomrape, mistletoe and dodder are included in the study of phytopathology. Dodder, for example, can be a conduit for the transmission of viruses or virus-like agents from a host plant to a plant that is not typically a host, or for an agent that is not graft-transmissible.

  • Cell wall-degrading enzymes: These are used to break down the plant cell wall in order to release the nutrients inside.
  • Toxins: These can be non-host-specific, which damage all plants, or host-specific, which cause damage only on a host plant.
  • Effector proteins: These can be secreted into the extracellular environment or directly into the host cell, often via the Type three secretion system. Some effectors are known to suppress host defense processes. This can include: reducing the plants internal signaling mechanisms or reduction of phytochemicals production. [14] Bacteria, fungus and oomycetes are known for this function. [4][15]
  • Spores: Spores of phytopathogenic fungi can be a source of infection on host plants. Spores first adhere to the cuticular layer on leaves and stems of host plant. In order for this to happen the infectious spore must be transported from the pathogen source, this occurs via wind, water, and vectors such as insects and humans. When favourable conditions are present, the spore will produce a modified hyphae called a germ tube. This germ tube later forms a bulge called an appressorium, which forms melanized cell walls to build up tugour pressure. Once enough turgor pressure is accumulated the appressorium asserts pressure against the cuticular layer in the form of a hardened penetration peg. This process is also aided by the secretion of cell wall degrading enzymes from the appressorium. Once the penetration peg enters the host tissue it develops a specialized hyphae called a haustorium. Based on the pathogens life cycle, this haustorium can invade and feed neighbouring cells intracellularly or exist intercellulary within a host. [16]

Some abiotic disorders can be confused with pathogen-induced disorders. Abiotic causes include natural processes such as drought, frost, snow and hail flooding and poor drainage nutrient deficiency deposition of mineral salts such as sodium chloride and gypsum windburn and breakage by storms and wildfires. Similar disorders (usually classed as abiotic) can be caused by human intervention, resulting in soil compaction, pollution of air and soil, salinisation caused by irrigation and road salting, over-application of herbicides, clumsy handling (e.g. lawnmower damage to trees), and vandalism. [ citation needed ]

Epidemiology: The study of factors affecting the outbreak and spread of infectious diseases. [17]

A disease tetrahedron (disease pyramid) best captures the elements involved with plant diseases. This pyramid uses the disease triangle as a foundation, consisting of elements such as: host, pathogen and environment. In addition to these three elements, humans and time add the remaining elements to create a disease tetrahedron.

History: Plant disease epidemics that are historically known based on tremendous losses:

- Chestnut blight in North America [20]

Factors affecting epidemics:

Host: Resistance or susceptibility level, age and genetics.

Pathogen: Amount of inoculum, genetics, and type of reproduction

Plant disease resistance is the ability of a plant to prevent and terminate infections from plant pathogens.

Structures that help plants prevent disease are: cuticular layer, cell walls and stomata guard cells. These act as a barrier to prevent pathogens from entering the plant host.

Once diseases have over come these barriers, plant receptors initiate signalling pathways to create molecules to compete against the foreign molecules. These pathways are influenced and triggered by genes within the host plant and are susceptible to being manipulated by genetic breeding to create varieties of plants that are resistant to destructive pathogens. [21]

Domestic quarantine Edit

A diseased patch of vegetation or individual plants can be isolated from other, healthy growth. Specimens may be destroyed or relocated into a greenhouse for treatment or study.

Port and border inspection and quarantine Edit

Another option is to avoid the introduction of harmful nonnative organisms by controlling all human traffic and activity (e.g., the Australian Quarantine and Inspection Service), although legislation and enforcement are crucial in order to ensure lasting effectiveness. Today's volume of global trade is providing—and will continue to provide—unprecedented opportunities for the introduction of plant pests. [McC 1] In the United States, even to get a better estimate of the number of such introductions, and thus the need to impose port and border quarantine and inspection, would require a substantial increase in inspections. [McC 2] In Australia a similar shortcoming of understanding has a different origin: Port inspections are not very useful because inspectors know too little about taxonomy. There are often pests that the Australian Government has prioritised as harmful to be kept out of the country, but which have near taxonomic relatives that confuse the issue. And inspectors also run into the opposite - harmless natives, or undiscovered natives, or just-discovered natives they need not bother with but which are easy to confuse with their outlawed foreign family members. [BH 1]

X-ray and electron-beam/E-beam irradiation of food has been trialed as a quarantine treatment for fruit commodities originating from Hawaii. The US FDA (Food and Drug Administration), USDA APHIS (Animal and Plant Health Inspection Service), producers, and consumers were all accepting of the results - more thorough pest eradication and lesser taste degradation than heat treatment. [22]

Cultural Edit

Farming in some societies is kept on a small scale, tended by peoples whose culture includes farming traditions going back to ancient times. (An example of such traditions would be lifelong training in techniques of plot terracing, weather anticipation and response, fertilization, grafting, seed care, and dedicated gardening.) Plants that are intently monitored often benefit from not only active external protection but also a greater overall vigor. While primitive in the sense of being the most labor-intensive solution by far, where practical or necessary it is more than adequate.

Plant resistance Edit

Sophisticated agricultural developments now allow growers to choose from among systematically cross-bred species to ensure the greatest hardiness in their crops, as suited for a particular region's pathological profile. Breeding practices have been perfected over centuries, but with the advent of genetic manipulation even finer control of a crop's immunity traits is possible. The engineering of food plants may be less rewarding, however, as higher output is frequently offset by popular suspicion and negative opinion about this "tampering" with nature.

Chemical Edit

Many natural and synthetic compounds can be employed to combat the above threats. This method works by directly eliminating disease-causing organisms or curbing their spread however, it has been shown to have too broad an effect, typically, to be good for the local ecosystem. From an economic standpoint, all but the simplest natural additives may disqualify a product from "organic" status, potentially reducing the value of the yield.

Biological Edit

Crop rotation may be an effective means to prevent a parasitic population from becoming well-established, as an organism affecting leaves would be starved when the leafy crop is replaced by a tuberous type, etc. Other means to undermine parasites without attacking them directly may exist.

Integrated Edit

The use of two or more of these methods in combination offers a higher chance of effectiveness.

Plant pathology has developed from antiquity, starting with Theophrastus, but scientific study began in the Early Modern period with the invention of the microscope, and developed in the 19th century. [23]


Chapter 8.4 : Processing Specimens for Fungal Culture

When a specimen is suspected to contain a fungal etiologic agent, it should be processed for fungal culture, regardless of direct microscopic findings. Recovery of fungal pathogens in culture provides definitive diagnosis of mycotic disease, identifies the etiologic agent of infection, and allows evaluation of in vitro susceptibility to antifungal agents. If there is insufficient material for both microscopy and culture, all of the specimen should be used for culture, since this is the more sensitive procedure for detection of fungi. Methods of specimen processing and culture are designed to retain the viability of the fungus and to obtain the maximum yield of organisms from clinical specimens. The choice of media for the isolation of fungi from clinical material is based primarily on the most likely species to be found in a particular site or under a recognized clinical condition. Selective media are included when other microorganisms, particularly bacteria, might also be present in the specimen. Specimens should be processed as soon as possible after receipt. Some specimens may require pretreatment prior to culture.


Identification and Pathogenicity of Fungal Pathogens Associated with Stem End Rots of Avocado Fruits in Kenya.

In Kenya, avocado (Persea americana Mill.) is one of the most important perennial tropical fruit crops and a major foreign exchange earner. In 2017, it accounted for about 74% by value of the total fruits exported from the country [1]. Currently, "Hass" avocado contributes approximately 80% of the avocado fruits produced and exported from Kenya [2]. Other cultivars produced include "Fuerte," "Puebla," "Duke," and "G6" [3]. Avocado production in Kenya is dominated by smallholder farmers (85%) within several agroecological zones, who mainly produce for the export market, and the remainder is sold in the local markets. Seventy percent (70%) of the avocado fruits are produced in the central and eastern regions of the country. The fruits are exported mainly to the European Union [2,4]. Since the year 2000, the acreage under avocado production has increased significantly, leading to increased export of the avocado fruit from Kenya [4]. The increased production is fueled by high demand for avocado fruits in the global market due to consumer awareness of the dietary value of the fruit [5]. Despite the increased production and export of avocado fruits from Kenya, high incidences of postharvest fungal diseases, including anthracnose and SER, limit marketing of the fruits and contribute to increased losses by the producers [6, 7].

The symptoms of stem end rot (SER) develop on the avocado fruit as it ripens. It is characterized by shriveling, followed by brown to black rot that starts at the stem end of the fruit. As the rot progresses, internal vascular bundles may have black to brown colorations and eventually the whole fruit is consumed by the rot [8, 9]. Fruits hardly display SER symptoms before harvest. Furthermore, SER often occur at the packing house during transit or after marketing.

Various fungal species have been reported to cause SER on avocado fruits. In Chile, the fungal pathogens reported to cause SER included members of Botryosphaeriaceae family, namely Diplodia mutila, D. pseudoseriata, D. seriata, Dothiorella iberica, Lasiodiplodia theobromae, Neofusicoccum australe, N. nonquaesitum, and N. parvum [10]. In Italy, N. parvum, Colletotrichum gloeosporioides, or C. fructicola and Diaporthe foeniculacea or D. sterilis were the most isolated SER pathogens [9]. In California, Neofusicoccum luteum and Phomopsis perseae were reported [8] while in South Africa, Thyronectria pseudotrichia, Dothiorella aromatica, Pestalotiopsis versicolor, Lasiodiplodia theobromae, Rhizopus stolonifer, Fusarium sambucinum, and Fusarium solani were reported [11].

In Kenya, however, the actual pathogen causing SER has not been identified, but on the other hand, anthracnose pathogens have been described [12]. Therefore, this study aimed at identifying the fungal pathogen associated with SER of avocado fruits in the central highlands of Kenya and testing their pathogenicity.

2.1. Study Area and Sample Collection. The study was conducted in Murang'a County, which is the leading county in production and export of avocado fruits in Kenya [1]. Geographically, the county lies between latitudes 0[degrees]34' south and 1[degrees]07' south and longitudes 36[degrees] east and 37[degrees]27' east, with an elevation of 914 m a.s.l in the east and 3,353 m a.s.l in the west. Avocado fruits are cultivated in the agroecological zones two, three, and four that have 18.0[degrees]C to 27.2[degrees]C average temperature ranges and 1600mm-900mm average annual rainfall [13].

Between September 2017 and March 2018, systematic sampling was used to select 162 orchards included in the study. The orchards had more than five "Hass" avocado fruit trees. Six mature avocado fruits were harvested at random from each five randomly selected avocado fruit trees in every sampled orchard. In addition, 10 "Hass" fruits, at different stages of ripening, were bought from different traders in three major markets (Kandara, Kirwara, and Maragwa) within the county at weekly intervals for two months. A total of 453 fruits from 4,860 fruits harvested from the orchards and 240 fruits from the market were sampled, packed in cartons, and transported to Kenya Agricultural and Livestock Research Organization (KALRO), Kandara, where they were incubated at room temperature (22[degrees]C-25[degrees]C) for 7-14 days to allow development of SER.

2.2. Fungal Isolation. The 207 fruits from the orchards and 125 fruits from the market that displayed SER symptoms were washed with clean tap water, surface-sterilized with 2% sodium hypochlorite for one minute, rinsed in distilled water, and air-dried. Small pieces of flesh from the margins of symptomatic flesh were placed aseptically in 9 cm diameter Petri dishes containing potato dextrose agar (PDA) amended with streptomycin sulfate and incubated at room temperature (22[degrees]C-25[degrees]C) for five days. Pure cultures were obtained by transferring the mycelia tips on 1.5% (wt/vol) water agar (WA) and allowed to grow overnight. Hyphal tips of the mycelia growth in the WA were later transferred onto PDA amended with streptomycin sulfate. Slant universal bottle was used to preserve the pure cultures of the pathogen and stored in the fridge at 4[degrees]C for later use.

2.2.1. Preparation of Conidial Suspension. Fourteen-day-old pure cultures in PDA were flooded with sterile distilled water. A sterile wire loop was used to scrape off the conidia and bring them to suspension. The suspension was filtered through a double-layer muslin cloth and the collected filtrate diluted serially to 1 * [10.sup.5]. A haemocytometer was used to adjust the spore concentration.

2.3. Morphological Characterization of the Isolate. To induce conidia production, small pieces of mycelia from the isolates were transferred into 9 cm diameter Petri dishes with PDA amended with autoclaved avocado wood chips and incubated at 25 [+ or -] 1[degrees]C for four weeks. The isolates were morphologically identified based on cultural and microscopic characteristics as described by Valencia et al. [10], Phillips et al. [14], and Watanabe [15]. Lactophenol blue was used in microscopic identification. The length and width of conidia (N = 50) from each isolate were measured using light microscope ZeissPrimo Star, coupled to AxioCam ERc 5s camera.

2.4. Molecular Characteristics

2.4.1. DNA Extraction. An improved fungal extraction protocol described by Innis et al. [16] was used to extract DNA from three representative isolates of each species. Pure fungal cultures derived from the single spores incubated in PDA were used. Forty milligram (mg) of mycelium was placed in a microcentrifuge tube containing 300 [micro]l of extraction buffer (Tris-HCl, 200 mM Ph 8.5 EDTA, 25 mM 1 M NaCl 250 mM SDS, 0.5%) with glass beads. The tubes were placed in a fastprep[R]-24 genogrinder for one minute at 2000 rpm. Two hundred microlitre ([micro]l) of 3 mM sodium acetate pH5.2 was added and refrigerated at -20[degrees]C for 10 minutes. After incubation, the samples were centrifuged for 10 minutes at 13000 rpm. After that, the supernatants were transferred into fresh 1.5 ml microcentrifuge tubes. Equal amounts of isopropanol were added to the supernatants and allowed to stand for five minutes at room temperature. After five minutes, the samples were centrifuged for 10 minutes at 13000 rpm and the supernatant was discarded. Five hundred [micro]l of 70% ethanol was then added to the pellets and centrifuged at 13000 rpm for 10 minutes to wash the pellet. The nucleic acid pellets obtained were air-dried and then resuspended in 50 [micro]l of low salt TE buffer (Tris-HCl, 1 mM, pH 8 EDTA, 0.1 mM) and stored at -20[degrees]C for later use. The quality of DNA was determined by agarose gel electrophoresis and quantified using a NanoDrop ND-1000Spectrophotometer. DNA was standardized or normalized to 20 ng/[micro]l for polymerase chain reactions (PCR).

2.4.2. DNA Amplification and Sequencing. The extracted DNA was used as templates in PCR. Two sets of primers, ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCC TCC GCT TAT TGA TAT GC), ITS5 (GGA AGT AAA AGT CGT AACAAG G) and ITS1, were used in the amplification of the internal transcribed region rDNA of the fungal isolates [16]. PCR reaction volumes of 25 [micro]l containing 2.5 [micro]l of 0.2 [micro]M of each primer, 5 * My Taq reaction buffer, 0.25 [micro]l Taq polymerase (Bioline, Meridian Life Science, Memphis, USA), 40 ng/[micro]l of each DNA template, and 12.75 [micro]l of molecular water were used. For amplification, the GeneAmp 9700 DNA Thermal Cycler (Perkin-Elmer) was used. The process involved an initial denaturing step at 94[degrees]C for 30 s, followed by 35 cycles, denaturing at 94[degrees]C for 30 s, annealing at 55[degrees]C for 30 s followed by extension for 1 minute at 68[degrees]C, and a final extension step of 5 min at 68[degrees]C. To confirm amplification, the PCR products were run on 1.5% agarose gel and visualized under UV light using ENDURO[TM] GDS. The PCR products were cleaned using the Qiagen PCR cleaning kit according to manufacturer instructions and submitted for Sanger sequencing with forward and reverse primers at Inqaba Africa Genomic platform, South Africa.

2.4.3. Bioinformatics Analysis. Sequence data was analyzed by assigning reads to samples, indexes, primers, and adapters. The primers were marked using Picard (https://broadinstitute.github. io/picard/index.html). Bam2fastq (https://gsl.hudsonalpha.org/ information/software/bam2fastq) was used to convert the resultants' bam files to fastq. The overall sequencing quality of the reads was evaluated visually using the Fast QC program (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/). The quality parameters used in filtering the reads included a minimum length of 250 bp and a minimum QC value of 30. Trimming was done corresponding to the adapters and low-quality sequences from all the reads. Subsequent analysis and processing of the reads was done in the CLC Genomic Workbench 11.0, where the overlapping reads were merged. The de novo assembly of the unassembled reads and the raw reads' alignment was performed using CLC Genomics Workbench 11.0 with default parameters (minimum contig = 100 bp, 23 K-mer, similarity fraction = 80%, and length fraction = 50%). BLAST analysis of the ITS sequences was done to support the morphological identification of the samples at the NCBI database. The ITS sequences were deposited in GeneBank using BankIT.

2.5. Pathogenicity Test. To establish Koch's postulates, Geotrichum candidum, Fusarium equiseti, Fusarium oxysporum, Fusarium solani, Lasiodiplodia theobromae, Neofusicoccum parvum, and Nectria pseudotrichia were subjected to pathogenicity test as described by Freeman et al. [17] and Twizeyimana et al. [8]. Healthy "Hass" fruits were harvested from the farms known to have a low incidence of SER within Murang'a County. The fruits were washed with clean tap water to remove any soil debris. The fruits were surface-sterilized by dipping in 75% ethanol for about three minutes, rinsed with distilled water, and then air-dried. Each of the isolates was subjected to two methods of inoculation.

A sterile cork borer (5 mm diameter) was used to wound the stem end of each fruit and mycelial discs of equivalent diameter obtained from the edge of actively growing pure cultures were placed on the wound. Six inoculated fruits for each pathogen and six control fruits inoculated with plain PDA were arranged on individual trays and covered with cling film to conserve moisture and avoid contamination. The fruits were incubated at room temperature of 24[degrees]C [+ or -] 1.

After snapping the pedicel of air-dried fruits, conidial suspension (5 * [10.sup.-5] conidial/ml) was placed on stem end opening and covered with cling film. Six inoculated fruits for each pathogen and six control fruits inoculated with distilled water were arranged in individual trays and covered with cling film. The inoculated fruits were incubated at 24 [+ or -] 1[degrees]C. Evaluation was done after 12 days by cutting the fruits longitudinally and rating SER symptoms on a 0-4 rating scale as follows: 0 = no visible rot 1 = 1-25% rot 2 = 25-50% rot 3 = 50-75% rot 4 = [greater than or equal to] 75% rot (Figure 1). At the end of the pathogenicity test, reisolation from the symptomatic fruits was made, and reisolated fungal colonies compared morphologically to the original isolates [8, 9]. SER severity on avocado fruits was calculated using the following formula [18]:

percent disease index (PDI) = sum of numerical ratings/no. of fruits examined * maximum grade * 100. (1)

2.6. Data Analysis. The Sanger sequenced data was analyzed and processed using CLC Genomic Workbench version 11.0. Analysis of Variance (ANOVA) followed by Tukey's post-hoc which was used to compare the mean percentage growth rate of inoculated fungi, while Student's t-test was used to compare SER lesions on fruits under different methods of inoculation. Statistical analysis was performed using Min tab v8 (Minitab, LLC).

3.1. Isolation and Identification of Fungal Species. After incubation of the fruits for 7-14 days, dark brown to black rot developed on the avocado fruits, and fungal mycelia were occasionally observed on the fruit surface. Internally, a discoloration of the vascular bundles was observed (Figure 2). As the fruit ripened, the rot progressed on the whole fruit. A total of 207 isolates were collected from the fruits from the orchards, and 125 isolates were collected from fruits from the markets.

Based on colony and conidial features the isolates were grouped into seven groups (Table 1).

Group 1 Lasiodiplodia theobromae colony on PDA was round and smooth. At first, white aerial filamentous mycelia with grey center developed. With age, colony turned grey and then dark grey to black (Figures 3(a) and 3(b)). The pycnidia were grey in colour and either simple or aggregated. The conidia were subovoid to ellipsoid. Initially, they were aseptate, thick-walled, and hyaline however, with time, they formed a single medium septum and became dark brown ranging from 17.35 to 29.31 * 11.23 to 14.91 [micro]m (mean 22.68 * 5.70 [micro]m). The morphological characteristics were consistent with what was described by Valencia et al. [10], Phillips et al. [14], and Watanabe [15].

Group 2 Neofusicoccum parvum colony on PDA was rough with irregular margins. Initially, white dense filamentous aerial mycelia developed and turned from dark to black with time (Figures 3(c) and 3(d)). Pycnidia were black, globose, and simple or aggregated. The conidia were bluntly round to subovoid, aseptate, and hyaline with granular content, and with time they turned from light brown to black with a size of 19.77 to 15.25 * 4.10 to 7.5 [micro]m (mean 17.01 * 5.70 [micro]m). The morphological characteristics were consistent with what was described by Valencia et al. [10] and Phillips et al. [14].

Group 3 Nectria pseudotrichia colonies on PDA were white, cottony, with filamentous, aerial mycelia growth. The colony growth was regular and rough, with smooth margins (Figures 3(e) and 3(f)). The conidia were ovoid to subovoid with greenish granular content ranging from 6.27 to 12.50 * 2.20 to 9.40 [micro]m (mean 8.49 * 4.95 [micro]m). The morphological characteristics were consistent with what was described by Hirooka et al. [19].

Group 4 Fusarium solani colonies on PDA were white, cottony, with floccose mycelium. The colony margins were regular and smooth. The rate of growth was low. The underside was pale to brown in colour (Figures 3(g) and 3(h)). The microconidia were hyaline, oval and some were cylindrical with smooth edges ranging from 5.02 to 8.52 * 2.91 to 5.50 [micro]m (mean 6.88 * 3.79 [micro]m), while the macroconidia were hyaline, slightly curved, and broad with two to three septa reaching within 13.05 to 34.18 * 2.10 to 5.50 [micro]m (mean 18.85 * 3.36 [micro]m). The morphological characteristics were consistent with what was described by Hafizi et al. [20] and Watanabe [15].

Group 5 Fusarium oxysporum colonies on PDA were with abundant white to creamy aerial mycelia. The colony margins were smooth and sometimes slightly looped. The reverse side of the colony was pale red to peach violet in colour (Figures 3(i) and 3(j)). Numerous ovoid to kidney-shaped microconidia without septa of 11.2 to 19.9 * 4.5 to 8.4 [micro]m (mean 15.4 * 6.1 [micro]m) were produced. The macroconidia were thin-walled, falcate to almost straight, and both ends were almost pointed with 2-3 septa ranging from 22.1 to 43.9 * 5.1 to 12.5 [micro]m (mean 28.4 * 7.5 [micro]m). The characteristics were similar to what was observed by Hafizi et al. [20], Hussain et al. [21] and Watanabe [15].

Group 6 Fusarium equiseti colonies on PDA were white, with abundant cottony mycelium that browned with age. Pale to dark brown diffusible pigmentation was observed (Figures 3(k) and 3(l)). The microconidia were not present however, long and slender slightly curved at the ends with three to six septa macroconidia of 25.3 to 46.7 * 3.5 to 4.6 [micro]m (mean 37.2 * 3.24 [micro]m) were observed as similarly observed by Motlagh [22] and Watanabe [15].

Group 7 Geotricum candidum colonies on PDA were not dense and white to beige appressed onto culture medium with smooth margins (Figures 3(m) and 3(n)). The mycelia formed smooth margined arthroconidia, which were hyaline, one-celled, and subglobose or cylindrical with either rounded or truncated apices reaching 6.1 to 19.7 * 2.3 to 10.3 [micro]m (mean 11.38 * 5.56 [micro]m). The fungus morphological features were consistent with those described by Zhang et al. [23], Alam et al. [24], and Watanabe [15].

Further more, to support the morphological identification of the samples, molecular markers ITS5 and ITS4 and ITS1 and ITS5 were used for molecular identification and consistently yielded high levels of species discrimination. PCR amplification for the ITS yielded products of 526 to 550 bp. From the blast analysis, fungal isolates were able to identify seven species. The isolates reported in this study have been associated with tropical fruits. These included F. equiseti (MK922072, MK922069), F. oxysporum (MK922065), F. solani (MK922070, MK922071, MK922066), G. candidum (MK215811, MK922075), L. theobromae (MK922068, MK922073), N. parvum (MK922067), and N. pseudotrichia (MK922074). The closest match between isolates from this study and those mined from the GeneBank had a range from 99 to 100% similarity and are shown in (Table 2).

3.2. Pathogenicity Tests. The avocado fruits inoculated with mycelia and those inoculated with spore suspensions developed similar symptoms as observed in fruits obtained from orchards and markets (Figure 1). All the inoculated fruits developed SER symptoms regardless of the isolate or the method of inoculation used. However, disease severity differed across the different fungal species as well as the method of inoculation (Table 3). When inoculated with mycelia, SER severity ranged between 6.67% and 90.83% and when spore suspension was used the severity ranged between 97.50% and 16.67% (Table 3). Lasiodiplodia theobromae, N. parvum, and N. pseudotrichia caused the most severe SER symptoms in both inoculations, and they might be considered the most virulent. No symptoms were observed on the control fruits. Statistical differences (p < 0.05) were detected in symptoms development when "Hass" avocado fruits were differentially inoculated with either mycelia or conidial suspensions of N. parvum, N. pseudotrichia, F. solani, F. equiseti, F. oxysporum, and G. candidum. However, there was no statistical significance in symptoms development when inoculated with L. theobromae (Table 3).

We report that avocado SER was caused by Lasiodiplodia theobromae, Neofusicoccum parvum, Nectria pseudotrichia, Fusarium solani, Fusarium oxysporum, Fusarium equiseti, and Geotricum candidum in the central highlands of Kenya. This is the first report on identification of SER fungal pathogens of avocado fruits in Kenya. The identified pathogens have been associated with SER of avocado fruits in other avocado growing regions in the world such as North America (California), Chile, South Africa, and Italy [8-11]. From the current study, L. theobromae was the most frequently isolated pathogen, followed by N. parvum and N. pseudotrichia. The study corroborates reports by Galsurker et al. [25] that identified L. theobromae as an emerging pathogen of fruits SER worldwide. The pathogen has been associated with SER of mangoes and pawpaw [26, 27] and also identified as a major pathogen that causes postharvest disease of many fruits [28].

Further more, results corroborate findings from other avocado growing regions of the world where members of Botryosphaeriaceae family were reported to be the leading cause of SER of avocado fruits. Botryosphaeriaceae species have been reported to cause SER of avocado in South Africa, Italy, California, and New Zealand. In South Africa N. pseudotrichia was the most isolated pathogen, and occasionally, L. theobromae was isolated. In Italy, California, and New Zealand, N. parvum was the most isolated pathogen [8-11, 29]. Temperatures influence SER pathogen predominant in an area. Botryosphaeriaceae species thrive in high temperature, while water stress stimulates latent infections by the species [25, 30]. The avocado fruit production in Murang'a County is concentrated in the lower region of the county, characterized by warm weather and temperature ranges between 18.0[degrees]C and 27.2[degrees]C [13]. These could explain why L. theobromae and N. parvum were the most isolated fungal pathogens.

In California N. parvum and other species of Botryosphaeriaceae (N. australe, N. luteum, Fusicoccum aesculi, and Dothiorella iberica) were associated with SER [8]. However, in our study, only N. parvum was isolated, similar to reports on SER pathogen of avocado fruits in Italy [9]. Three Fusarium species, namely, F. solani, F. oxysporum, and F. equiseti, were found to be minor pathogens of SER in Kenya. Similar findings were reported from South Africa, New Zealand [11,31], and Ethiopia [30]. Geotricum candidum was exclusively isolated from four avocado fruits from the market and never from the orchard fruits. The pathogen has been associated with sour rots of tomatoes, citrus fruits, and vegetables [32]. In the open air markets in Kenya, where the avocado fruits were bought, fruits from avocado, citrus, and other species are placed together, thus allowing for cross-infection between those fruit species.

Colletotrichum gloeosporioides, which have been previously reported as causing SER of avocado in Italy and California [8, 11], was not isolated in our study corroborating reports from Chile [10]. Moreover, Twizeyimana et al. [8] identified C. gloeosporioides as a weak avocado SER pathogen and is only important when in combination with other SER pathogens.

Morphological characteristics together with DNA analysis were used to identify and differentiate L. theobromae, N. parvum, and N. pseudotrichia. Lasiodiplodia theobromae grew fast and colonised the Petri dish in two days. Neofusicoccum parvum and N. pseudotrichia colonized the Petri dish in four and five days, respectively. The three pathogens showed almost similar morphological features. However, ITS sequences of these fungi clearly allowed the differentiation of the species.

Lasiodiplodia theobromae was the most isolated pathogen from fruits from both orchards and markets, followed by N. parvum and N. pseudotrichia. During pathogenicity studies, the three pathogens also caused the most severe SER on avocado fruits. The three pathogens are, therefore, identified as the main causal agents of avocado SER in Kenya.

Further studies should be conducted in other avocado growing regions in the country to get a clear picture of SER etiology in Kenya. Besides, preharvest and postharvest SER management practices of avocado fruits in the country should be established.

The data used to support the findings of this study are included in the article.

The authors declare that there are no conflicts of interest regarding the publication of this paper.

The authors are grateful to the management of Kenya Agricultural and Livestock Research Organization (KALRO), Kandara and Biotechnology Research Centre, Kabete, for providing the necessary facilities to carry out morphological and molecular studies. The authors also acknowledge Inqaba Biotech., South Africa, for DNA sequencing.

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E. K. Wanjiku, (1,2) J. W. Waceke, (1) B. W. Wanjala [ID], (3) and J. N. Mbaka (4)

(1) Department of Agriculture Science and Technology, Kenyatta University (KU), Nairobi, Kenya

(2) Department of Animal Health and Production, Mount Kenya University (MKU), Nairobi, Kenya

(3) Biotechnology Research Institute, Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya

(4) Horticulture Research Institute, Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya

Correspondence should be addressed to B. W. Wanjala [email protected]

Received 29 November 2019 Revised 5 June 2020 Accepted 11 June 2020 Published 9 July 2020

Academic Editor: Giuseppe Comi

Caption: Figure 1: Symptoms of SER on artificial inoculated "Hass" avocado fruits. Guide to severity scoring of the disease rot scale (0-4). (a) and (b) represent 0 (c) and (d) represent 1 (e) and (f) represent 2 (g) and (h) represent 3 (i) and (j) represent 4.

Caption: Figure 2: SER symptoms displayed after incubation. (a) brown discoloration of the fruit pulp (b) black discoloration of the vascular bundles (c) and (d) fungal mycelia developing on the surface.

Caption: Figure 3: Characteristics of colony (reverse and front) of pathogenic isolates of SER on PDA. (a) and (b) represent reverse and front of Lasiodiplodia theobromae (GA11) (c) and (d) Neofusicoccumparvum (GA7) (e) and (f) Nectriapseudotrichia (GA13) (g) and (h) Fusarium solani (1GEF8) (i) and (j) Fusarium oxysporum (MS4a) (k) and (l) Fusarium equiseti (1GF17) (m) and (n) Geotricum candidum (GA6).


Volume 8 - 2017 - Index

18. Neophyllachora gen nov. (Phyllachorales), three new species of Phyllachora from Poaceae and resurrection of Polystigmataceae (Xylariales)
Dayarathne MC, Maharachchikumbura SSN, Jones EBG, Goonasekara ID, Bulgakov TS, Al-Sadi AM, Hyde KD, Lumyong S and McKenzie EHC
Mycosphere 8(10), 1598–1625

20. The edible wide mushrooms of Agaricus section Bivelares from Western China
Zhang MZ, Li GJ, Dai RC, Xi YL, Wei SL and Zhao RL
Mycosphere 8(10), 1640–1652

24. Novel fungal species of Phaeosphaeriaceae with an asexual/sexual morph connection.
Karunarathna A, Papizadeh M, Senanayake IC, Jeewon R, Phookamsak R, Goonasekara ID, Wanasinghe DN, Wijayawardene NN, Amoozegar MA, Shahzadeh Fazeli SA, Camporesi E, Hyde KD, Weerahewa HLD, Lumyong S, McKenzie EHC
Mycosphere 8(10), 1818–1834

26. Establishment of Zygosporiaceae fam. nov. (Xylariales, Sordariomycetes) based on rDNA sequence data to accommodate Zygosporium.
Li JF, Phookamsak R, Jeewon R, Tibpromma S, Maharachchikumbura SSN, Bhat DJ, Chukeatirote E, Lumyong S, Hyde KD, McKenzie EHC
Mycosphere 8(10), 1855–1868

27. Can ITS sequence data identify fungal endophytes from cultures? A case study from Rhizophora apiculata
Doilom M, Manawasinghe IS, Jeewon R, Jayawardena RS, Tibpromma S, Hongsanan S, Meepol W, Lumyong S, Jones EBG, Hyde KD
Mycosphere 8(10), 1869–1892

56. Magnicamarosporium diospyricola sp. nov. (Sulcatisporaceae) from Thailand
Phukhamsakda C, Bhat DJ, Hongsanan S, Tibpromma S, Yang JB, Promputtha I
Mycosphere 8(4) 512–520

60. New record of Trichoglossum rasum from Asia
Prabhugaonkar A, Pratibha J
Mycosphere 8(4) 583–591

62. Taxonomy and phylogeny of Sparticola muriformis sp. nov. on decaying grass
Karunarathna A, Phookamsak R, Wanasinghe DN, Wijayawardene NN, Weerahewa HLD, Khan S, Wang Y
Mycosphere 8(4) 603–614

66. Mycosphere notes 1-50: Grass (Poaceae) inhabiting Dothideomycetes
Thambugala KM, Wanasinghe DN, Phillips AJL, Camporesi E, Bulgakov TS, Phukhamsakda C, Ariyawansa HA, Goonasekara ID, Phookamsak R, Dissanayake A, Tennakoon DS, Tibpromma S, Chen YY, Liu ZY, Hyde KD
Mycosphere 8(4) 697–796

68. Diaporthe species associated with peach tree dieback in Hubei, China
Dissanayake AJ, Zhang W, Liu M, Hyde KD, Zhao W, Li XH, Yan JY
Mycosphere 8(5) 533–549

70. Molecular phylogenetic analysis reveals seven new Diaporthe species from Italy
Dissanayake AJ, Camporesi E, Hyde KD, Zhang Wei, Yan JY, Li XH
Mycosphere 8(5) 853–877

71. The current status of species in Diaporthe
Dissanayake AJ, Phillips AJL, Hyde KD, Yan JY, Li XH
Mycosphere 8(5) 1106–1156

90. Genetic variability in Setchelliogaster tenuipes (Setch.) Pouzar based on DNA barcoding ITS
Sulzbacher MA, Bevilacqua CB, Baldoni DB, Jacques RJS, Antoniolli ZI et al.
Mycosphere 8(7) 899–907

93. Mycosphere Essays 19: Recent advances and future challenges in taxonomy of coelomycetous fungi
Wijayawardene NN, Papizadeh M, Phillips AJL, Wanasinghe DN, Bhat DJ, Weerahewa HLD, Shenoy BD, Wang Y, Huang YQ
Mycosphere 8(7) 934–950

94. Mycorrhizal fungi status in organic farms of south Florida
Toprak B, Soti P, Jovel E, Alverado L, Jayachandran K
Mycosphere 8(7) 951–958

102. Tar spot fungi from Thailand
Tamakaew N, Maharachchikumbura SSN, Hyde KD, Cheewangkoon R.
Mycosphere 8(8) 1054–1058

110. New Cylindrocladiella spp. from Thailand soils
Lombard L, Cheewangkoon R, Crous PW
Mycosphere 8(8) 1088–1104

120. Towards a natural classification of Amplistromataceae
Daranagama DA, Tian Q, Liu XZ, Hyde KD
Mycosphere 8(9) 1392–1402

121. Powdery mildew species on papaya – a story of confusion and hidden diversity
Braun U, Meeboon J, Takamatsu S, Blomquist C, Fernández Pavía SP, Rooney-Latham S, Macedo DM
Mycosphere 8(9), 1403–1423

122. Mycosphere Essay 19. Cordyceps species parasitizing hymenopteran and hemipteran insects
Shrestha B, Tanaka E, Hyun MW, Han JG , Kim CS , Jo JW , Han SK , Oh J, Sung JM, Sung GH
Mycosphere 8(9), 1424–1442

124. Towards incorporating asexual fungi in a natural classification: checklist and notes 2012–2016
Wijayawardene NN, Hyde KD, Tibpromma S, Wanasinghe DN, Thambugala KM, Tian Q, Wang Y, Fu L
Mycosphere 8(9), 1457–1555

125. New species and records of Bipolaris and Curvularia from Thailand
Marin-Felix Y, Senwanna C, Cheewangkoon R and Crous PW
Mycosphere 8(9), 1556–1574

126. Dendryphiella fasciculata sp. nov. and notes on other Dendryphiella species
Liu NG, Hongsanan S, Yang J, Lin CG, Bhat DJ, Liu JK, Jumpathong J, Boonmee S, Hyde KD and Liu ZY
Mycosphere 8(9), 1575–1586

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