Fungal Infestation Research Articles

Plant organ abscission and the green island effect caused by a coleopteran’s gall on Miconia cf cinnamomifolia (Melastomataceae): larval survival and mortality factors

Galls are characterized by inducing cellular differentiation (hyperplasia or hypertrophy) resultant of the action of some organisms, mainly insects, on structures/organs of their host plants. The galls cause physiological changes in host plants, altering host traits, and their growth and survival. The early abscission of galled plant organs can be a form of plant defense. Therefore, the galls decayed more slowly than the healthy leaves, or the surrounding healthy leaf tissue in some abscised galled leaves, forming “green island galls”. This study reported an instance where the host plant Miconia cinnamomifolia (Melastomataceae) abscises leaves galled by an unidentified coleopteran’s gall on the soil of a fragment of Atlantic Forests, Brazil. Once on the forest soil galls were exposed to a new set of potential natural enemies, as pathogens and predators. Consequently, larval survival decrease of 79% to 36% in four months and fungal infestation increase of 2% to 21%. Neither size nor weight of the galls differed between categories of mortality factors and larval survival. It was discussed the adaptive nature of the “Green Island Effect” as a counter-response of gallers to leaf abscission, which is a known plant defense strategy.

Traditional maize post-harvest management practices amongst smallholder farmers in Guatemala

Much of the maize that is produced in Guatemala is planted, harvested and handled via subsistence-oriented agricultural practices, strongly connected to Mayan heritage. This post-harvest assessment study was done to characterize the current practices used in the region of Huehuetenango, Guatemala, in order to identify the different grain handling practices in the region as well as possible factors contributing to post-harvest losses of maize. A total of 280 families representing 14 rural communities were surveyed through interviews. Survey revealed that most (88%) of interviewed farmers prefer to dry the maize cobs after harvest by laying them in stacks exposed to direct sunlight. After drying, harvested maize is stored until consumption along with purchased maize kernels from the market. Among storage practices, 62% of surveyed families store the maize as shelled kernels; while 38% store it on cobs. When storing shelled maize, bags are the preferred containers among 81% of farmers, while only 14% use metal silos. Among farmers who stored maize on cobs, 74% use the tapanco as the preferred storage structure. Forty-one percent of farmers indicated storing the maize for at least 4 months. During the storage time, 61% of farmers perform grain quality checks once a week. Moreover, 65% perform pest control during storage; however, in most cases, the control is not preventive but corrective. For 49% of farmers, the main cause of loss between harvest and consumption is the mishandling of grain moisture, leading to insect and fungal infestation. With this data, it was possible to identify diverse maize harvesting, drying, storage and consumption practices within the studied communities. Understanding the traditional post-harvest practices will help better design intervention steps to improve these practices and to increase food security and food safety for smallholder farmers in the Guatemalan Highlands.

Incidence of Eosinophilia in Rural Population in North India: A Study at Tertiary Care Hospital

BACKGROUND- Eosinophilia is abnormally high number of eosinophils in the blood. Normally, eosinophils constitute 1 to 6% of the peripheral blood leukocytes, at a count of 350 to 650 per cubic millimeter. Eosinophilia can be categorized as mild (less than 1500 eosinophils per cubic millimeter), moderate (1500 to 5000 per cubic millimeter), or severe (more than 5000 per cubic millimeter). Eosinophilia may be primary or secondary. The aim of the study was to determine the incidence of eosinophilia and evaluate the patients thoroughly for the cause of eosinophilia. METHOD- The study was conducted in the Pathology department in the medical college and hospital in rural are of Punjab. Complete blood count and peripheral blood film study was done in almost all the patients visiting the hospital. The patients with eosinophilia were segregated and were made to fill the detailed proforma. The information included family history, chief complaints, food habits, disease history and drug history. A thorough general examination and diagnostic work up followed. RESULT- In all 3442 (10.7%) patients visiting the hospital had eosinophilia; out of this 2136 (62%) patients had mild eosinophilia, 1297 (37.7%) had moderate and 9 (0.3%) had severe eosinophilia. 2451(71.2%) patients were males and 991 (28.8%) were females. Age group of patients varied from 3 yrs to 74 yrs. The chief complaints of our patients were related to fever, cough, rashes, breathlessness, wheezing etc. other major group of patients had symptoms of abdominal pain, diarrhea, pallor, pica and loss of appetite. CONCLUSION- Eosinophilia appears to be a common occurrence in the in the rural population around our hospital. The most common etiologies were due to parasitic, protozoal or fungal infestations and infections. Allergy related problem also constituted a significant health problem in the local population. DOI: 10.21276/apalm.2017.1044

Fluctuation in seed abundance has contrasting effects on the fate of seeds from two rapidly germinating tree species in an Asian tropical forest.

The seed predator satiation hypothesis states that high seed abundance can satiate seed predators or seed dispersers, thus promoting seed survival. However, for rapidly germinating seeds in tropical forests, high seed abundance may limit dispersal as the seeds usually remain under parent trees for long periods, which may lead to high mortality due to rodent predation or fungal infestations. By tracking 2 species of rapidly germinating seeds (Pittosporopsis kerrii, family Icacinaceae; Camellia kissi, family Theaceae), which depend on dispersal by scatter-hoarding rodents, we investigated the effects of seed abundance at the community level on predation and seed dispersal in the tropical forest of Xishuangbanna Prefecture, Southwest China. We found that high seed abundance at the community level was associated with delayed and reduced seed removal, decreased dispersal distance and increased pre-dispersal seed survival for both plant species. High seed abundance was also associated with reduced seed caching of C. kissi, but it showed little effect on seed caching of P. kerrii. However, post-dispersal seed survival for the 2 plant species followed the reverse pattern. High seed abundance in the community was associated with higher post-dispersal survival of P. kerrii seeds, but with lower post-dispersal survival of C. kissi seeds. Our results suggest that different plant species derive benefit from fluctuations in seed production in different ways.

Prospects and Problems of Dietary Glucosinolates in Animal Feeding

Brassica originated feed and fodders are the chief source of glucosinolates in animal diets. High intake of glucosinolates induce several health and production problem in animals, however low glucosinolates containing meals may be good source of protein especially of sulfur containing amino acids. Recent research on glucosinolates shown that feeding of low glucosinolates containing meals reduce fungus infestation of ration, increase isothiocynate content of animal produce and increased peripheral fat with higher contents of unsaturated fatty acids (C18:00, 1 trans, C22:2) in carcass and milk fat, are advantages of Brassica meal feeding because isothiocyantes and unsaturated fatty acids exerts anti-carcinogenic properties. The deleterious effects of high glucosinolate containing meals could be eliminated using a suitable detoxification technology and keeping the level of glucosinolate within tolerable limits.

SEED MYCOFLORA OF Lens culinaris MEDDIK. FROM DIFFERENT STORAGE CONDITIONS AND IN-VITRO INHIBITION USING PLANT EXTRACTS

Nine hundred sixty seeds of lentils (Lens culinaris) from three different storage sources were tested for seed fungi using Potato Dextrose Agar (PDA) and Blotter technique. Thirty five different fungi were isolated, PDA method being better than Blotter technique. Seeds from traditional village storage were found to be more infested with fungi than source local market and source Nepal Agricultural Research Centre (NARC). The fungal infestation on seeds from village storage was 49.37%, source NARC was 44.37% and source local market was 30.31%. Aspergillus flavus, Aspergillus niger, Alternaria alternata, Penicillium chrysogenum and Fusarium oxysporum were the fungi having higher incidence and frequency. Surface disinfection by 2% NaOCl reduced the fungal incidence and frequency. The aqueous extract of three plants viz, Ageratum houstonianum, Eclipta prostrata and Vitex negundo at 25%, 50% and 100% concentration were assessed in-vitro for antifungal activity against Alternaria alternata, Aspergillus niger, Chaetomium funicola and Fusarium oxysporum. Among these fungi, Alternaria alternata was best controlled by Ageratum houstonianum; Aspergillus niger by Eclipta prostrata; Fusarium oxysporum by Vitex negundo and Ageratum houstonianum. The inhibition increased from lower concentration (25%) to higher (100%).ECOPRINT 23: 19-27, 2016

Occurrence of Fusarium Mycotoxins in Cereal Crops and Processed Products (Ogi) from Nigeria.

In Nigeria, maize, sorghum, and millet are very important cash crops. They are consumed on a daily basis in different processed forms in diverse cultural backgrounds. These crops are prone to fungi infestation, and subsequently may be contaminated with mycotoxins. A total of 363 samples comprising of maize (136), sorghum (110), millet (87), and ogi (30) were collected from randomly selected markets in four agro-ecological zones in Nigeria. Samples were assessed for Fusarium mycotoxins contamination using a multi-mycotoxin liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Subsequently, some selected samples were analysed for the occurrence of hidden fumonisins. Overall, 64% of the samples were contaminated with at least one toxin, at the rate of 77%, 44%, 59%, and 97% for maize, sorghum, millet, and ogi, respectively. Fumonisins were the most dominant, especially in maize and ogi, occurring at the rate of 65% and 93% with mean values of 935 and 1128 μg/kg, respectively. The prevalence of diacetoxyscirpenol was observed in maize (13%), sorghum (18%), and millet (29%), irrespective of the agro-ecological zone. Other mycotoxins detected were deoxynivalenol, zearalenone, and their metabolites, nivalenol, fusarenon-X, HT-2 toxin, and hidden fumonisins. About 43% of the samples were contaminated with more than one toxin. This study suggests that consumption of cereals and cereal-based products, ogi particularly by infants may be a source of exposure to Fusarium mycotoxins.

Copper and aluminium deposition by cold-plasma spray on wood surfaces: effects on natural weathering behaviour

Atmospheric pressure plasma was used to deposit thin layers of copper and aluminium micro-particles on the surface of Scots pine sapwood (Pinus sylvestris L.) boards. Three different loadings of metal particles were established. Additional wood boards were topcoated with a commercial acrylic binder. Boards were exposed to natural weathering for 18 months. Discolouration of copper-treated boards was slowed down, and the treatment at highest loading displayed the best appearance. Aluminium treatment was not sufficient to prevent or reduce discolouration. The application of an acrylic binder as topcoating enhanced the general appearance of metal-treated boards. Evaluation of treated boards did not reveal any reduction in crack formation or water uptake due to the particle deposition. Infrared spectroscopy suggested that copper does not protect lignin from photo-degradation. Nevertheless, copper treatment reduced fungal infestation on wood; at highest copper loading, blue stain did not penetrate through the treated surface.

Farmers Perception on Fungicide Use for Developing Sustainable Environment and Conservation of Biodiversity: A Study in Cooch Behar District of India

Fungicide was essential for control of infestation of fungus on different crop. Farmers were use different type of fungicide on crop field. Their perception on fungicide use may be different. Different government and private organisation took initiative to aware the farmer about the use of fungicide. But still a problem was found by Cooch Behar Krishi Vigyan Kendra that farmer perception of fungicides use were not homogeneous and the different sources of information may be use by the farmers for fungicide application. So a study was conducted at Gopalpur village in Cooch Behar district, West Bengal, India to know the farmer perception on fungicide use and sources of information utilize for fungicide application. The data were collected during June, 2016. The research design was followed in the study was survey research method. The sample size of the study was 100.The dependent variable of this study was perception and independent variables were age, gender, number of family member, caste, land holding, education, annual income and sources of information. The descriptive statistics like frequency, percentage, Pearson’s product moment correlation and other statistical tools were used for the investigation. This study had shown the relation of the perception of fungicide use with the different independent variable and the role of different extension mechanism for fungicide information providing. It was concluded from the study that farmers’ perception of fungicide use were not homogeneous and the different socio-economic variable may affect the perception of the farmers on fungicide use. You can submit your Manuscripts at: https://symbiosisonlinepublishing.com/submitManuscript.php

English

Practices used by smallholder dairy farmers for handling of feeds at the farm pose a risk of mycotoxins to dairy animals and dairy products, hence a public health concern. The aim of the study was to document the on-farm practices of handling feeds used by these farmers and how they influence the growth of mycotoxin producing fungi together with prevailing extrinsic conditions. Study involved the use of structured questionnaire for interview of smallholder dairy farmers (n=120) for on-farm feed handling practices and collection of feed samples (n=97) for microbial analysis of the mycotoxin producing molds. The fungi counts were interrelated with the feed handling practice and therefore a measure of its impact. Results found out that rural dairy system was characterized by practice of free range grazing unlike peri-urban system practice that had semi-intensive stall feeding. At the farm level, the type feeds storage facility and the type and condition of feeds were found to be significant risk factors (p<0.05) for infestation of mycotoxic fungi. Feed contamination on farm at the sub-value chains with mycotoxic fungi is primarily due to poor storage facilities exposing feed to environmental conditions that favors fungi growth.   Key words: Feeds, fungi, mycotoxins, Aspergillus, Fusarium, Kenyan smallholder farmers.

Molecular Divergence and Identification of Aspergillus Species in Dry Fishes of Gujarat, India

The fungal infestations have become significant cause for the quality and economic loss. They often cause infections to the handlers and consumers. Some of the fungal species are potent allergens, mycotoxin producers and can even cause superficial and pulmonary infection. Reports of previous studies are restricted to genus level due to overlapping of morphological characters. So, the present study was carried out to identify the predominant fungal species prevalent in fishes of west coast region by molecular technique. Totally, 24 sundried fishes were collected from the Veraval region of Gujarat and analyzed for the presence of fungal species. Randomly collected fungal strain was initially indentified based on morphological characters and polymerase chain reaction was carried out for the amplification of 18S rDNA gene. The amplicon was sequenced and analyzed using BLAST. The sequence analysis revealed that Aspergillus flavus, A. nidulans, A. oryzae, A. sydowii, A. rugulosa, A. niger, A. foetidus, Lichtheimia ramosa and Curvularia lunata were present in dried fish. The phylogenetic tree was constructed based on DNA sequence dissimilarity and revealed that two clades were formed among the fungal species with one clade belonging to A. nidulans, A. rugulosa and A. sydowii and the other to A. flavus and A. oryzae. Therefore, it is concluded that, in west coast region A. flavus is a dominant species observed in dry fish which is potent aflatoxin producers and highly carcinogenic in nature. So, handlers need stringent hygienic practices with suitable processing and packaging technique to control the fungal contamination.

Response of moth Plodia interpunctella to volatiles of fungus-infected and uninfected wheat grain

The aim of the study was to reveal volatile organic compounds (VOCs) produced by wheat grain, both before and after fungi infestation, which evoke reactions in Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), mated females. Solid phase micro-extraction (SPME) technique was used to collect volatiles and gas-chromatography, mass spectrometry (GC-FID/EAD, GC-MS) analysis and synthetic standards were applied to reveal and identify active compounds. Olfactory receptors of P. interpunctella females responded to five VOCs: 1-hexanol, nonanal, phenylacetaldehyde and 4-oxophorone (emitted by fungi-uninfected grain), and 3-methyl-1-butanol (emitted by fungi-infected grain). Behavioral tests carried out in olfactometers revealed attractive property of 1-hexanol and nonanal at low concentrations and repellent properties of 4-oxoisophorone to mated P. interpunctella females. Phenylacetaldehyde was neither an attractive nor repellent. 3-Methyl-1-butanol acted as an attractant at low concentrations and as repellent at higher ones. The latter compound could serve as a kairomone and allomone due to its different effects on behavior at higher and lower concentration.

Enhanced yeast feeding following mating facilitates control of the invasive fruit pest Drosophila suzukii

Summary The highly invasive spotted wing Drosophila Drosophila suzukii is a key pest of soft fruit and berries in Europe and North America, and development of control techniques is an urgent research challenge. Drosophila suzukii is widely associated with the yeast Hanseniaspora uvarum. Yeasts are symbionts of drosophilid flies and communicate with insects through volatile metabolites for spore dispersal. Accordingly, yeasts and behaviour‐modifying chemicals produced by yeasts are prospective tools for environmentally sound insect management. We first bioassayed flight attraction, feeding and oviposition of D. suzukii females in response to H. uvarum yeast and blueberries, which are a preferred host fruit. We then investigated the combined effect of yeast and insecticide on adult female oviposition behaviour and mortality towards the development of a yeast‐based control method. Following mating, attraction of female flies to blueberry and yeast odour cues was strongly enhanced. Yeast feeding significantly increased in mated females, while yeast did not increase oviposition on blueberries. This observation suggests that mated flies become attracted to yeast for feeding and to fruit for egg laying. A combined feeding–oviposition assay demonstrated different roles and interference between yeast and fruit stimuli: during the day after mating, females laid fewer eggs when yeast was available. The post‐mating yeast‐feeding response is an opportunity for the development of an attract‐and‐kill technique for population control of D. suzukii. Exposing flies to a blend of yeast and insecticide reduced oviposition and greatly enhanced adult fly mortality compared with an insecticide treatment alone. Synthesis and applications. Mated females are the key life stage for Drosophila suzukii population control. Egg‐laying females perforate fruit skin and fungal infestations ensue, even when eggs and larvae are killed off by insecticide sprays. Behaviour‐modifying chemicals, including yeast metabolites, enable environmentally safe insect management via manipulation of olfactory‐mediated reproductive behaviour. Our results highlight that yeast and yeast semiochemicals hold potential for D. suzukii management and that response modulation to olfactory stimuli following mating is a vital element for the development of D. suzukii control methods. Yeast feeding is enhanced in mated D. suzukii females, and this change in post‐mating behaviour can be exploited by an attract‐and‐kill strategy, combining a fly‐associated yeast with an insecticide. Furthermore, using the D. suzukii yeast mutualist, H. uvarum, may reduce non‐target effects and increase species specificity, which further contributes to the development of an efficient and safe control method.

Peat biomass degradation: Evidence from fungal and faunal activity in carbonized wood from the Eocene sediments of western India

An integration of reflected light and transmitted light study of Eocene lignite along with Scanning Electron Microscope (SEM) examination of carbonized wood from Kapurdi lignite of the Barmer Basin has yielded abundant fungal components and arthropod appendages. The carbonized wood section under SEM shows tyloses, a physiological process as a response to fungal infection. Scolecodonts and annelid body parts were also recovered from the studied sediments, indicating the peat biomass was a thriving habitat for detritivores. In petrographic examination of the lignite beds which underlay and overlay the carbonized wood section, the funginite is found to be associated with resinite, indicating that the funginite inclusion was not accidental but encapsulation of fungi by exuded resin of the plant as a defense mechanism. Funginite is found in higher percentages in detrohuminite (attrinite) groundmass in lignites. The degradation of the humic matter is attributed to the fungal infestation as well as faunal activity in the peat mire. This fungal–faunal interaction resulted in alteration of the organic matter and origin of macerals belonging to inertinite group.

Seasonal Variation of Indigenous Saccharomyces cerevisiae Strains Isolated from Vineyards of the Western Cape in South Africa

There is strong support for the use of naturally-occurring Saccharomyces cerevisiae strains that improve the sensory quality of wines and reflect the characteristics of a given region. Contrary to popular belief, S. cerevisiae is found at very low numbers on healthy, undamaged grapes and is rarely isolated from intact berries. The majority of studies on the population kinetics and geographic distribution of indigenous S. cerevisiae strains have not adequately focused on the variation in their numbers over a longer period of time. This paper discusses the results obtained in the first phase of a comprehensive research programme aimed at assessing how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Indigenous strains of S. cerevisiae were aseptically isolated from eight sites in four areas in the coastal regions of the Western Cape, South Africa, during 1995 through 1998. Thirty colonies per site were isolated and the S. cerevisiae strains were characterised by electrophoretic karyotyping. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns in 1998 resulted in a gradual recovery of the indigenous population. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. Commercial wine yeast strains were recovered in only a few instances and the likelihood that commercial yeasts will eventually replace the natural yeast microflora in vineyards seems remote.

Effects of an aphid pest on an invasive plant in the Peruvian Andes

An exotic aphid pest (Aphis cytisorum) has infested an invasive plant (Spartium junceum) in the Peruvian Andes since 2005. In turn, the aphids facilitated the spread of sooty mold fungus (Capnodium sp.) that covers the surface of ephemeral leaves and stems. We evaluated the infestation of aphids and fungus on S. junceum and on another Andean plant species in 2007 and 2015, measuring the effects of infestation on reproductive output of S. junceum in 2008. Individuals of S. junceum were heavily infested by aphids and fungus and close to half of populations were dead in the early years of infestation, although mortality had decreased over time. Infested plants produced larger fruits and seeds, but approximately a quarter of the production of uninfested plants. Viability and germination of seeds produced by infested plants were similar to those produced by uninfested plants. Aphis cytisorum was monophagous on S. junceum, although there is a risk that it can eventually infest other Andean plant species, because this aphid is polyphagous elsewhere. The strong impact of aphid/fungus infestation on survival and reproduction of S. junceum suggest that initially these natural enemies could have worked as a biological control. However, the population of S. junceum persists and shows evidence of repopulation, indicating the ineffectiveness of biocontrol. Our results support the importance of a long-term monitoring to determine the effectiveness of biocontrol agents and provide critical information for future related studies, considering that plant pests and disease infestations will likely increase under changing climate.

Effect of microwave radiation on seed viability, survival of Aspergillus niger van Tieghem and oil quality of oilseeds crops canola, soybean and safflower

The effect of microwave's radiation on seed viability of three different oilseed crops, spores of Aspergillus niger and quality of extracted oil from treated seeds over various exposure times was evaluated. The seeds were exposed to 2450 MHz. at five different power levels of 0, 100, 200, 400, 600 and 800 W for two exposure times of three and five minutes. At a given time, a direct negative relationship between seed viability and microwave's radiation power level was detected. Substantial variation in the lethality of tested seeds to microwave's power levels was apparent in the fiducial limits of the estimated LD50 values in probit analysis approach. A similar trend of A. niger spores’ susceptibility to microwave radiation was detected. The microwaves' radiation and exposure time did not impact one another and a significant interaction was not detected. Short term fungal infection did not cause substantial quantitative and qualitative damage to the oilseeds. The oil quality was generally unaffected by microwave radiation and fungal infestation for tested oilseeds. Moreover, microwave radiation decreased seed germination percentage and vigor index. The microwave radiation could provide an effective and friendly environmental treatment technique for improving the dietary consumption of the oil in any seed disinfestation program.

Effects of sawdust amendment on forest soil fungal community and infestation by cockchafers

Effects of sawdust amendment on forest soil fungal community and infestation by cockchafers

Mycobiota in the brood cells of the European beewolf, Philanthus triangulum (Hymenoptera: Crabronidae)

Mass provisioning insects have to cope with microbial spoilage of their food supplies. As their fitness is directly linked to the availability of high quality food for their offspring, they have evolved various mechanisms for preserving these resources. The European beewolf, Philanthus triangulum, uses several mechanisms to not only preserve the food for their larvae, paralyzed workers of the European honeybee, Apis mellifera, but also protect the larvae that develop on the stored food. To assess the spectrum of fungi that pose a threat to beewolf brood cells, we manipulated brood cells by removing the insect defenses. We monitored the subsequent fungal infestations that would have been prevented by the beewolf defense mechanisms and isolated and identified the mold fungi. The cosmopolitan and highly competitive species of Aspergillus, in particular A. flavus, dominated the mold in beewolf brood cells. All other infestations could also be attributed to generalist mold fungi that are commonly found in soil and also on insects. Our findings indicate that beewolf brood cells can be colonized by a broad range of opportunistic soil mold fungi. Thus, it seems highly adaptive that beewolves employ general, broad spectrum defense mechanisms.

Alfalfa hay induced primary photosensitization in horses

Photosensitization, also known as photodermatitis, occurs when phototoxic or photoactive substances accumulate in the skin and interact with sunlight to result in an often severe, crusting, itching or painful dermatitis in unpigmented and/or lightly haired areas of the skin. Primary photosensitization, caused by direct ingestion of photosensitizing agents, has been reported anecdotally in horses after ingestion of alfalfa hay. Between 2004 and 2014, several large outbreaks of primary photosensitization in horses fed primarily alfalfa hay were investigated in California. Alfalfa hay samples were collected and carefully examined for the presence of known photosensitizing plants and pesticide residues but none were identified. Select hay samples were evaluated for unusual fungal infestation and for phototoxicity assay using a specific Candida albicans assay; results were negative. In the 2004 outbreak, a feeding study was conducted with three horses exclusively fed alfalfa hay that was suspected to have caused the outbreak. Two weeks after ingestion of alfalfa hay, two horses developed several lesions in non-pigmented skin characterized as chronic ulcerative and necrotizing dermatitis with superficial vasculitis, which was consistent with photosensitization. In the 2014 outbreak, seven different implicated alfalfa hay samples were analyzed for chlorophyll a and b, and pheophorbide a. These compounds had been suspected to play a role in alfalfa-induced primary photosensitization. The chlorophyll contents ranged from 0.90 to 2.30 mg/g in the alfalfa hay samples, compared to 1.37 and 2.94 mg/g in locally grown alfalfa and orchard grass hay. The pheophorbide a levels ranged from 3.36 to 89.87 µg/g in alfalfa samples compared to 81.39 and 42.33 µg/g in control alfalfa and orchard grass hay samples. These findings eliminate chlorophyll a, chlorophyll b, and pheophorbide a as possible causes for alfalfa-hay induced primary photosensitization.